Surfactant lipids, compositions thereof, and uses thereof

ABSTRACT

The invention generally relates to methods to inhibit inflammation or pathogen infection by administering at least one anionic lipid or compositions comprising at least one anionic lipid to an individual. The invention also relates to methods to prevent or inhibit respiratory syncytial virus (RSV) infection by administering at least one anionic lipid or compositions comprising at least one anionic lipid to an individual. The invention further relates to compositions comprising randomly mixed surfactant lipids and methods to produce the compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 12/057,967, filed Mar. 28, 2008, now U.S. Pat. No. 8,367,643 whichclaims the benefit of priority under 35 U.S.C. §119(e) of U.S.Provisional Application Nos. 60/908,837, filed Mar. 29, 2007, and61/025,298, filed Jan. 31, 2008, the entire disclosures of which areincorporated herein by reference for all purposes.

GOVERNMENT SUPPORT

This invention was made with government support under NIH Grant Nos.HL045286 and HL073907, each awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention generally relates to methods to inhibit inflammation orpathogen infection by administering at least one anionic lipid orcompositions comprising at least one anionic lipid to an individual. Theinvention also relates to methods to prevent or inhibit respiratorysyncytial virus (RSV) infection by administering at least one anioniclipid or compositions comprising at least one anionic lipid to anindividual. The invention further relates to compositions comprisingrandomly mixed surfactant lipids and methods to produce thecompositions.

BACKGROUND OF THE INVENTION

Pulmonary surfactant was initially identified as a lipoprotein complexthat reduces surface tension at the air-liquid interface of the alveolarcompartment of the lung (Pattle, R. E. 1955. Nature 175:1125; Clements,J. A. 1957. Proc Soc Exp Biol Med 95:170). Pulmonary surfactant issynthesized and secreted by alveolar type II cells (King et al., 1973.Am J Physiol 224:788). Approximately 10% of surfactant is composed ofproteins, including the hydrophilic surfactant proteins A and D (SP-Aand SP-D), and the hydrophobic proteins, SP-B and SP-C (Kuroki andVoelker. 1994. J. Biol. Chem. 269:25943). SP-A and SP-D are nowrecognized to play important roles in innate immunity (Sano and Kuroki.2005. Mol Immunol 42:279). SP-A and SP-D directly interact with variousmicroorganisms and pathogen-derived components (Lawson and Reid. 2000.Immunol Rev 173:66). Moreover, by associating with cell surfacepattern-recognition receptors, SP-A and SP-D regulate inflammatorycellular responses such as the release of lipopolysaccharide(LPS)-induced proinflammatory cytokines (Sano et al., 1999. J. Immunol.163:387). LPS, derived from Gram-negative bacteria, is a potentstimulator of inflammation (O'Brien et al., 1980. J Immunol 124:20;Ulevitch and Tobias. 1995. Annu Rev Immunol 13:437). LPS molecules areengaged by the plasma LPS binding protein (LBP) (Wright et al., 1990.Science 249:1431) and transferred to CD14, aglycosylphosphatidylinisitol (GPI)-anchored protein, abundantlyexpressed on macrophages. LPS responses are dependent on theperipherally associated plasma membrane protein MD-2 (Nagai et al. 2002.Nat Immunol 3:667). and the membrane-spanning complex formed bytoll-like receptor (TLR) 4 (Poltorak et al., 1998. Science 282:2085),through which signaling is propagated. TLRs activate four intracellularprotein kinase cascades, the IB kinase (IKK)/NF-kB transcription factorcascade, the extracellular signal-regulated kinase (ERK), c-JunNH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase(MAPK) cascades, leading to the induction of many key cytokine genesthat are essential for the innate immune response (Takeda et al., 2003.Annu Rev Immunol 21:335; Medzhitov, R. 2001. Nat Rev Immunol 1:135;Barton and Medzhitov. 2003. Science 300:1524). At least one importantfunction of SP-A and SP-D is to suppress the inflammatory response ofthe lung to LPS.

By weight, approximately 90% of surfactant consists of lipids. Althoughthe lipid composition varies in different species, its major componentis phosphatidylcholine (PC) (70-80%) of which nearly 80% is disaturated,consisting primarily of dipalmitoyl-phosphatidylcholine (DPPC). Inaddition, pulmonary surfactant contains variable amounts ofphosphatidylglycerol (PG) (7-18%), phosphatidylinositol (PI) (2-4%) andphosphatidylethanolamine (PE) (2-3%) (Veldhuizen et al. 1998. BiochemBiophys Acta 1408:90). In contrast to PC, more than 50% of PG isunsaturated in many species, and in humans there is little or nodisaturated PG (Schmidt et al., 2002. Am J Physiol Lung Cell Mol Physiol283:1079; Wright et al., 2000. J Appl Physiol 89:1283). The functions ofthe minor phospholipid and the neutral lipid components of surfactantare largely unclear and there is a need in the art for furtherinformation regarding the roles of these components.

Previous work has provided some evidence that specific phospholipids canmodulate inflammation. Oxidized phospholipid inhibits LPS-inducedinflammatory responses in human umbilical-vein endothelial cells(Bochkov et al., 2002. Nature 419:77). Dioleoyl-phosphatidylglycerol(DOPG) inhibits phospholipase A2 secretion via a downregulation of NF-kBactivation in guinea pig macrophages (Wu et al. 2003. Am J Respir CritCare Med 168:692). Treponemal membrane phosphatidylglycerol inhibitsLPS-induced immune responses from macrophages by inhibiting the bindingof biotinylated LPS to LBP and blocked the binding of soluble CD14(sCD14) to LPS (Hashimoto et al., 2003. J Biol Chem 278:44205).Cardiolipin, PG and PI exhibit an inhibitory effect on LPS-induced TNF-αproduction by human macrophages, most likely by a blockade of thebinding of LPS aggregates to LBP (Mueller et al., 2005. J Immunol172:1091). However, very few reports have focused on the potentialanti-inflammatory roles of surfactant phospholipids on either alveolaror non-alveolar macrophages. Moreover, the relationship betweensurfactant phospholipids and CD14 or other pattern recognition receptorshas not been clearly identified.

Various studies have made connections between surfactant PG content anddisease. For example, in idiopathic pulmonary fibrosis patients, somegroups reported decreased unsaturated PG in surfactant (Veldhuizen etal., 1998, Biochem Biophys Acta 1408:90; Honda et al., 1988, Lung166:293; and Saydain et al., 2002, Am J Resp Crit Care Med 166:839). Inanother disease, ARDS, Schmidt et. al. have reported significantreduction in the unsaturated PG recovered in BALF (Schmidt et al., 2001,Am J Respir Crit Care Med 163:95). The issues of cause and effect in theabove diseases remain unclear.

LPS is a major cause of acute lung injury (ALI) and acute respiratorydistress syndrome (ARDS) (Atabai and Matthay. 2002. Thorax 2002;Rubenfeld et al., 2005. N Engl J Med 353:1685). ALI/ARDS is alife-threatening condition in which inflammation of the lungs andaccumulation of fluid in the alveoli leads to low blood oxygen levels.Over a period of 25 years the annual incidence of ALI/ARDS is 335,000,with 147,000 deaths per year. The most common risk factor for ALI wassevere sepsis with a suspected pulmonary source (46%), followed bysevere sepsis with a suspected nonpulmonary source (33%).

Given the severity of symptoms associated with many inflammatoryconditions, including those affecting the respiratory system, there is acontinued need for agents useful in controlling inflammation and therebypreventing and/or treating conditions or diseases associated withinflammation.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a method to inhibit inflammationor pathogen infection, comprising administering to an individual whohas, or is at risk of developing said inflammation or pathogeninfection, an amount of at least one anionic lipid or related compound,wherein the amount of the anionic lipid or related compound is effectiveto inhibit said inflammation or pathogen infection, and wherein theanionic lipid has the following characteristics: a hydrophobic portion,a negatively charged portion, and an uncharged, polar portion.

In some embodiments, the anionic lipid or related compound is selectedfrom the group consisting of: unsaturated phosphatidylglycerol,unsaturated phosphatidylinositol, saturated short chainphosphatidylglycerol, saturated short chain phosphatidylinositol,anionic sphingolipid, anionic glycerolipid, unsaturatedlyso-phosphatidylglycerol, saturated lyso-phosphatidylglycerol,unsaturated lyso-phosphatidylinositol, and saturatedlyso-phosphatidylinositol, or a derivative thereof.

In some embodiments, the anionic lipid is selected from the groupconsisting of: an unsaturated phosphatidylglycerol, an unsaturatedphosphatidylinositol, a saturated short chain phosphatidylglycerol, anda saturated short chain phosphatidylinositol, or a derivative of theanionic lipid.

In some embodiments, the inflammation or pathogen infection isassociated with a toll-like receptor (TLR) selected from the groupconsisting of: TLR1, TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, and TLR10.

In some embodiments, the inflammation or pathogen infection isassociated with a toll-like receptor (TLR) selected from the groupconsisting of: TLR1, TLR2, TLR3, TLR6, TLR7, TLR8, and TLR10.

In some embodiments, the individual has a bacterial infection associatedwith TLR1.

In some embodiments, the individual has an infection, condition, ordisease associated with TLR2 selected from the group consisting of:cytomegalovirus infection, herpes simplex virus infection, measles, aprotozoan infection, a fungal infection, and Varicella zoster infection.

In some embodiments, the individual has an infection, condition, ordisease associated with TLR3 selected from the group consisting of: aviral infection (such as rhinovirus infection or parainfluenza virusinfection) and a cancer.

In some embodiments, the individual has an infection, condition, ordisease associated with TLR6 selected from the group consisting of: abacterial infection, a protozoan infection and a fungal infection.

In some embodiments, the individual has an infection, condition, ordisease associated with TLR7 selected from the group consisting of: anautoimmune disease, a cancer, and a viral infection.

In some embodiments, the viral infection is selected from the groupconsisting of: human immunodeficiency virus infection, rhinovirusinfection, parainfluenza virus infection, human parechorvirus infection,influenza infection, papilloma virus infection, and Varicella zosterinfection.

In some embodiments, the individual has an infection, condition, ordisease associated with TLR8 selected from the group consisting of:autoimmune disease, basal cell carcinoma, Bowen's disease, condyloma,genital warts, human immunodeficiency virus (HIV), rhinovirus,parainfluenza virus, Human parechovirus, melanoma, and molluscacontagiosa.

In some embodiments, the individual has a respiratory disorder.

In some embodiments, the respiratory disorder is selected from the groupconsisting of: adult respiratory distress syndrome (ARDS), acute lunginjury (ALI), viral infection associated with asthma, chronicobstructive pulmonary disease (COPD), pneumonia, bronchitis,tuberculosis, reactive airway disease syndrome, interstitial lungdisease, rhinitis, and parasitic lung disease.

Another aspect of the invention relates to a method to prevent orinhibit viral infection, comprising administering to an individual whohas, or is at risk of developing a viral infection, at least one anioniclipid or related compound, wherein the amount of the anionic lipid orrelated compound is effective to prevent or inhibit said viralinfection, and wherein the anionic lipid has the followingcharacteristics: a hydrophobic portion, a negatively charged portion,and an uncharged, polar portion.

In some embodiments, the anionic lipid or related compound is selectedfrom the group consisting of: unsaturated phosphatidylglycerol,unsaturated phosphatidylinositol, saturated short chainphosphatidylglycerol, saturated short chain phosphatidylinositol,anionic sphingolipid, anionic glycerolipid, unsaturatedlyso-phosphatidylglycerol, saturated lyso-phosphatidylglycerol,unsaturated lyso-phosphatidylinositol, and saturatedlyso-phosphatidylinositol, or a derivative thereof.

In some embodiments, the anionic lipid is selected from the groupconsisting of: an unsaturated phosphatidylglycerol, an unsaturatedphosphatidylinositol, a saturated short chain phosphatidylglycerol, anda saturated short chain phosphatidylinositol, or a derivative of theanionic lipid.

Another aspect of the invention relates to a method to prevent orinhibit respiratory syncytial virus (RSV) infection, comprisingadministering to an individual who has, or is at risk of developing aviral infection, at least one anionic lipid or related compound, whereinthe amount of the anionic lipid or related compound is effective toprevent or inhibit said RSV infection, and wherein the anionic lipid hasthe following characteristics: a hydrophobic portion, a negativelycharged portion, and an uncharged, polar portion.

In some embodiments, the anionic lipid or related compound is selectedfrom the group consisting of: unsaturated phosphatidylglycerol,unsaturated phosphatidylinositol, saturated short chainphosphatidylglycerol, saturated short chain phosphatidylinositol,anionic sphingolipid, anionic glycerolipid, unsaturatedlyso-phosphatidylglycerol, saturated lyso-phosphatidylglycerol,unsaturated lyso-phosphatidylinositol, and saturatedlyso-phosphatidylinositol, or a derivative thereof.

In some embodiments, the anionic lipid is selected from the groupconsisting of: an unsaturated phosphatidylglycerol, an unsaturatedphosphatidylinositol, a saturated short chain phosphatidylglycerol, anda saturated short chain phosphatidylinositol, or a derivative of theanionic lipid.

In some embodiments, the individual is a neonatal infant.

In some embodiments, the anionic lipid or related compound isadministered to the infant prior to any indication of infection withRSV.

In some embodiments, the anionic lipid or related compound isadministered to the infant subsequent to identification of a symptom ofor confirmation of infection of the infant with RSV.

In some embodiments, the anionic lipid or related compound isphosphatidylglycerol, or a derivative thereof.

In some embodiments, the anionic lipid or related compound ispalmitoyl-oleoyl-phosphatidylglycerol (POPG), or a derivative thereof.

In some embodiments, the anionic lipid or related compound isdimyristoyl-phosphatidylglycerol (DMPG), or a derivative thereof.

In some embodiments, the anionic lipid or related compound isunsaturated phosphatidylinositol, or a derivative thereof.

In some embodiments, the anionic lipid or related compound is an anionicsphingolipid or a derivative thereof.

In some embodiments, the anionic lipid or related compound is an anionicglycerolipid or a derivative thereof.

In some embodiments, the anionic lipid or related compound is anunsaturated or saturated lyso-phosphatidylglycerol, or a derivativethereof.

In some embodiments, the anionic lipid or related compound is anunsaturated or saturated lyso-phosphatidylinositol or a derivativethereof.

In some embodiments, the anionic lipid or related compound isadministered as a homogeneous lipid preparation.

In some embodiments, the anionic lipid or related compound isadministered as a composition comprising a homogeneous lipid preparationof the anionic lipid or related compound.

In some embodiments, the anionic lipid or related compound isadministered as a composition comprising a preparation of randomly mixedsurfactant lipids combined with a homogeneous lipid preparation of theanionic lipid or related compound.

In some embodiments, the anionic lipid or related compound isadministered as a preparation of randomly mixed surfactant lipids,wherein the anionic lipid or related compound comprises at least about50% of the total lipids in said randomly mixed surfactant lipids.

In some embodiments, the anionic lipid or related compound isadministered to the respiratory tract of the individual.

Another aspect of the invention relates to a method to inhibitinflammation, comprising administering to an individual who has, or isat risk of developing said inflammation, a composition comprising atleast one anionic lipid or related compound, wherein the anionic lipidor related compound is effective to inhibit said inflammation, andwherein the anionic lipid has the following characteristics: ahydrophobic portion, a negatively charged portion, and an uncharged,polar portion; and wherein the composition is selected from the groupconsisting of: a homogeneous lipid preparation consisting of the anioniclipid or related compound; a composition comprising a homogeneous lipidpreparation of the anionic lipid or related compound and at least oneagent for the treatment of inflammation; a composition comprising apreparation of randomly mixed surfactant lipids combined with ahomogeneous lipid preparation of the anionic lipid or related compound;and a preparation of randomly mixed surfactant lipids, wherein theanionic lipid or related compound comprises at least about 50% of thetotal lipids in said randomly mixed surfactant lipids.

In some embodiments, the anionic lipid or related compound is selectedfrom the group consisting of: unsaturated phosphatidylglycerol,unsaturated phosphatidylinositol, saturated short chainphosphatidylglycerol, saturated short chain phosphatidylinositol,anionic sphingolipid, anionic glycerolipid, unsaturatedlyso-phosphatidylglycerol, saturated lyso-phosphatidylglycerol,unsaturated lyso-phosphatidylinositol, and saturatedlyso-phosphatidylinositol, or a derivative thereof.

In some embodiments, the anionic lipid is selected from the groupconsisting of: an unsaturated phosphatidylglycerol, an unsaturatedphosphatidylinositol, a saturated short chain phosphatidylglycerol, anda saturated short chain phosphatidylinositol, or a derivative of theanionic lipid.

In some embodiments, the composition is a preparation of randomly mixedsurfactant lipids combined with a homogeneous lipid preparation of theanionic lipid or related compound.

In some embodiments, the composition is a homogeneous lipid preparationof the anionic lipid or related compound and at least one additionalagent for treating inflammation.

Another aspect of the invention relates to a method to produce asurfactant composition, comprising (a) providing a substantiallyhomogeneous lipid preparation of at least one anionic lipid or relatedcompound, wherein the anionic lipid has the following characteristics: ahydrophobic portion, a negatively charged portion, and an uncharged,polar portion; and (b) adding the preparation of (a) to a preparation ofrandomly mixed surfactant lipids.

In some embodiments, the preparation of (a) is in aqueous solution.

In some embodiments, the preparation of (b) is in aqueous solution.

In some embodiments, the preparation is gently mixed to avoidsignificant fusion or intermixing of lipids between lipid bilayers ormicelles in (a) and (b).

In some embodiments, the lipids in the preparation of (a) comprise atleast 1% of the total lipids in the composition.

In some embodiments, the anionic lipid or related compound isphosphatidylglycerol, or a derivative thereof.

In some embodiments, the anionic lipid or related compound ispalmitoyl-oleoyl-phosphatidylglycerol (POPG), or a derivative thereof.

In some embodiments, the anionic lipid or related compound isdimyristoyl-phosphatidylglycerol (DMPG), or a derivative thereof.

In some embodiments, the anionic lipid or related compound isunsaturated phosphatidylinositol, or a derivative thereof.

In some embodiments, the anionic lipid or related compound is an anionicsphingolipid or a derivative thereof.

In some embodiments, the anionic lipid or related compound is an anionicglycerolipid or a derivative thereof.

In some embodiments, the anionic lipid or related compound is anunsaturated or saturated lyso-phosphatidylglycerol, or a derivativethereof.

In some embodiments, the anionic lipid or related compound is anunsaturated or saturated lyso-phosphatidylinositol or a derivativethereof.

Another aspect of the invention relates to a surfactant compositioncomprising a mixture of (a) a preparation of randomly mixed surfactantlipids and (b) one or more substantially homogeneous lipid preparationsof at least one anionic lipid or related compound, wherein the anioniclipid has the following characteristics: a hydrophobic portion, anegatively charged portion, and an uncharged, polar portion; wherein thepreparation of (b) is added to the preparation of (a) to form acomposition in which there is no significant fusion or intermixing oflipids between lipid bilayers of (a) and (b).

In some embodiments, the lipids in the preparation of (b) comprise atleast 1% of the total lipids in the composition.

In some embodiments, the anionic lipid or related compound isphosphatidylglycerol, or a derivative thereof.

In some embodiments, the anionic lipid or related compound ispalmitoyl-oleoyl-phosphatidylglycerol (POPG), or a derivative thereof.

In some embodiments, the anionic lipid or related compound isdimyristoyl-phosphatidylglycerol (DMPG), or a derivative thereof.

In some embodiments, the anionic lipid or related compound isunsaturated phosphatidylinositol, or a derivative thereof.

In some embodiments, the anionic lipid or related compound is an anionicsphingolipid or a derivative thereof.

In some embodiments, the anionic lipid or related compound is an anionicglycerolipid or a derivative thereof.

In some embodiments, the anionic lipid or related compound is anunsaturated or saturated lyso-phosphatidylglycerol, or a derivativethereof.

In some embodiments, the anionic lipid or related compound is anunsaturated or saturated lyso-phosphatidylinositol or a derivativethereof.

Another aspect of the invention relates to a lipid compositioncomprising randomly mixed surfactant lipids, wherein at least 50% of thetotal lipids in the composition is comprised of one or more anioniclipids or related compounds, wherein the anionic lipid has the followingcharacteristics: a hydrophobic portion, a negatively charged portion,and an uncharged, polar portion.

In some embodiments, the anionic lipid or related compound isphosphatidylglycerol, or a derivative thereof.

In some embodiments, the anionic lipid or related compound ispalmitoyl-oleoyl-phosphatidylglycerol (POPG), or a derivative thereof.

In some embodiments, the anionic lipid or related compound isdimyristoyl-phosphatidylglycerol (DMPG), or a derivative thereof.

In some embodiments, the anionic lipid or related compound isunsaturated phosphatidylinositol, or a derivative thereof.

In some embodiments, the anionic lipid or related compound is an anionicsphingolipid or a derivative thereof.

In some embodiments, the anionic lipid or related compound is an anionicglycerolipid or a derivative thereof.

In some embodiments, the anionic lipid or related compound is anunsaturated or saturated lyso-phosphatidylglycerol, or a derivativethereof.

In some embodiments, the anionic lipid or related compound is anunsaturated or saturated lyso-phosphatidylinositol or a derivativethereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Anionic phospholipids inhibit inflammatory mediator productioninduced by LPS. Liposomes composed of sphingomyelin (SM),phosphatidylethanolamine (PE) dipalmitoyl-phosphatidylcholine ((DPPC),phosphatidylserine (PS), palmitoyl-oleoyl-phosphatidylglycerol (POPG)and phosphatidylinositol (PI) were formed by bath-sonication for 30 minat room temperature. LPS (10 ng/ml) and different concentrations ofphospholipids were added to monolayer cultures of differentiated U937cells (A), or primary rat alveolar macrophages (B). At 6 h afterstimulation, media were collected and secreted TNF-α levels weredetermined in U937 cultures. NO production was determined 24 h afterstimulating rat alveolar macrophages. LPS stimulation in the absence ofphospholipid was set as 100%. The data shown are the means±S.E. fromthree separate experiments with duplicate samples in each experiment.The average TNF-α production upon LPS stimulation was (8.0±0.54 ng/ml).The average NO production upon LPS stimulation was 12.17±0.27 μm.

FIG. 2. The inhibitory effect of phosphatidylglycerols on LPS-inducedinflammatory mediator production is molecular species specific. PGliposomes were formed by bath-sonication for 30 min at room temperature.LPS (10 ng/ml) and different concentrations of PG were added tomonolayer cultures of differentiated U937 cells (A) or rat alveolarmacrophages (B). Media TNF-α measurements were performed 6 h afterstimulation. Media NO measurements were performed 24 h afterstimulation. LPS stimulation without PG was set at 100%. The molecularspecies of PG shown on the graph are 8:0;dioctanoyl-phosphatidylglycerol, 12:0; dilauroyl-phosphatidylglycerol(DLPG), 14:0; dimyristoyl-phosphatidylglycerol (DMPG), 16:0;dipalmitoyl-phosphatidylglycerol (DPPG), 18:0;distearoyl-phosphatidylglycerol, 16:0/18:1;palmitoyl-oleoyl-phosphatidylglycerol (POPG). The data shown are themeans±S.E. from three separate experiments with duplicate samples ineach experiment. The average TNF-α production upon LPS stimulation was11.3±0.7 ng/ml. The average NO production upon LPS stimulation was10.1±0.6 μM.

FIG. 3. Homotypic PG containing liposomes are most effective inantagonizing LPS action in the presence of surfactant lipids. Surfactantlipid (SL) and POPG were dried under nitrogen, and hydrated at 37° C.for 1 h. (A) SL and POPG were mixed in organic solvents prior to dryingand hydrating and subsequently liposomes were produced. (B) SL and POPGwere made as independent populations of liposomes that were subsequentlymixed prior to macrophage treatment. 10 ng/ml of LPS and differentconcentrations of liposome mixtures were added to monolayer cultures ofdifferentiated U937 cells. 6 h after stimulation, media were collectedand TNF-α production was determined LPS stimulation without phospholipidwas set as 100%. The data shown are the means±S.E. from three separateexperiments with duplicate samples in each experiment. The average TNF-αproduction upon LPS stimulation was 7.0±0.2 ng/ml.

FIG. 4. POPG inhibits LPS induced MAPK and IkBα phosphorylation andMKP-1 expression. POPG liposomes (200 μg/ml) were added to monolayercultures of differentiated U937 cells that received either no treatmentor 10 ng/ml LPS. After incubating for the indicated time, cells werelysed using lysis buffer containing detergent, protease inhibitors andphosphatase inhibitors. Aliquots with 15 μg of protein from lysates wereseparated by SDS-PAGE and transferred onto nitrocellulose membranes. Theamount of phosphorylation was detected using phospho-specific antibodiesto p38MAPK, p42/p44 ERK, p46-p54 JNK and phosphorylated IkBα. Todetermine equal loading of proteins between samples, the membranes wereprobed with rabbit polyclonal p46 JNK, p42/p44 ERK, p38MAPK and IkBαantibodies. The expression of MKP-1 was detected with a polyclonal MKP-1antibody.

FIG. 5. Quantification of POPG inhibition of LPS induced signaling.Western blot analysis as described in FIG. 4 was performed three or fourtimes on separate samples and the intensity of phospho-p38,phospho-IkBα, phosphoERK, phospho JNK, phospho IkBα, total IkBα andMKP-1 was calculated using NIH Image J1.34 software. Significance—*:p<0.05, **: p<0.01, when compared between LPS and LPS with POPGstimulation.

FIG. 6. Molecular specificity in POPG action. Liposomes composed ofPOPC, DPPG or POPG were added to monolayer cultures of differentiatedU937 cells that received either no treatment or 10 ng/ml LPS, asindicated. After 30 or 60 min cells were lysed and 15 μg of cellularprotein from cultures was separated by SDS-PAGE and transferred ontonitrocellulose membranes. Phosphorylated and nonphosphorylated proteinswere detected as described in FIG. 4.

FIG. 7. Comparative quantification of lipid dependent antagonism of LPSsignaling. Western blot analysis was performed as described in FIG. 6 inthree independent experiments. The intensity of the phosphorylated (p38,ERK, JNK and IkBα) and nonphosphorylated (total IkBα and MKP-1) proteinsof interest was measured using NIH Image J1.34 software.Significance—*p<0.05, **p<0.01.

FIG. 8. POPG, DMPG and PI antagonize the effects of LPS on primary humanalveolar macrophages. Human alveolar macrophages were isolated fromhealthy volunteer BALF and plated onto a 96-well plate. Two days afterplating, 10 ng/ml of LPS and 20 μg/ml of phospholipids were added tomonolayer cultures of human alveolar macrophages. 6 h after stimulation,media were collected and TNF-α production was determined by ELISA. LPSstimulation without phospholipid was set at 100%. The data shown are themeans±S.E. from three separate experiments with duplicate samples ineach experiment. The average TNF-α secretion after LPS stimulation was30.7±15.1 ng/ml. Significance—**: p<0.01, when compared with LPSstimulation in the absence of POPG.

FIG. 9. Anionic phospholipids modulate lung inflammation induced byintratrachealy administered LPS. A mixture of LPS (1 μg) andphospholipids (30 μg) in 20 μl of PBS was sprayed into murine tracheausing a MicroSprayer™ aerosolizer. At 18 h after stimulation, lungs werelavaged via the trachea. TNF-α production (A) was determined by ELISA.The number of leukocytes was counted and differential cell counts (B)were determined from at least 300 cells on cytocentrifuged preparations.Mouse KC (C) and MIP-2 (D) secretion were determined using Quantikinekits (R&D System). The data shown are the means±S.E. from six to eightmice. Significance—*: p<0.05, **: p<0.01, when compared between LPS andLPS plus POPG.

FIG. 10. Anionic phospholipids modulate lung inflammation induced byintravenously administered LPS. Phospholipids were dried under nitrogenand hydrated, and liposomes were formed using a Liposofast™. Thephospholipids (50 μg) in 20 μl of PBS were sprayed into murine tracheausing a MicroSprayer™ aerosolizer. At the same time, LPS (50 μg) in 200μl of PBS was intravenously administered to mice. 3 h after stimulation,lungs were lavaged via the trachea. TNF-α production (A) was determinedby ELISA. The number of leukocytes was counted and differential cellcounts (B) were determined from at least 300 cells on cytocentrifugedpreparations. Mouse KC (C) and MIP-2 (D) levels were determined usingQuantikine kits (R&D System). The data shown are the means±S.E. for sixto eight mice. Significance—*: p<0.05, **: p<0.01, when compared betweenLPS and LPS plus POPG.

FIG. 11. Anionic phospholipids block BODIPY-LPS association withRAW264.7 macrophages. Liposomes were prepared by bath-sonication at roomtemperature for 30 min. RAW264.7 cells (10⁶/tube) were incubated eitherwithout or with 1 μg/ml BODIPY-LPS in the presence or absence ofliposomes (200 μg/ml) at 4° C. for 4 h. Subsequently the cells werewashed by centrifugation and the cell associated fluorescence wasquantified by FACScan. Panel A shows the primary data for incubation ofcells without or with LPS and incubation with LPS in the presence ofeither PI or POPG. In panel B the mean fluorescence intensity (MFI)ratio of cells plus LPS/cells without LPS is plotted against differentphospholipid treatments. Values shown in B are means±SE for threeindependent experiments with duplicate determinations in eachexperiment. Significance—* p<0.05, ** p<0.01

FIG. 12. CD14 binds to solid phase lipids. Phospholipids (1.25 nmole) in20 μl of ethanol were placed onto microtiter wells and the solvent wasevaporated. Nonspecific binding was blocked with 20 mM Tris buffer (pH7.4) containing 0.15 M NaCl, 5 mM CaCl₂ (in the upper panel) or 2 mMEGTA (in the lower panel), and 5% (wt/vol) BSA (buffer A). Varyingconcentrations of human CD14 in buffer A were added and incubated at 37°C. for 1 h. The binding of CD14 to phospholipids was detected usinganti-CD14 monoclonal antibody as described under “ExperimentalProcedures.” The data shown are the means±S.E. from three separateexperiments with duplicate samples in each experiment.

FIG. 13. PG Inhibits CD14 binding to solid phase LPS. (A) Various typesof PG were coated onto microtiter plates, and incubated with CD14 (1μg/ml) at 37° C. for 1 h. The binding of CD14 to PG was detected usinganti-CD14 monoclonal antibody, and the ELISA based absorbance of CD14bound to POPG was defined as 100%. Types of PG shown on the graph are:dilauroylphosphatidylglycerol (DLPG), dimyristoyl-phosphatidylglycerol(DMPG), dipalmitoylphosphatidylglycerol (DPPG), and 16:0/18:1;palmitoyl-oleoyl-phosphatidylglycerol (POPG). (B) LPS (2 μg) in 20 μl ofethanol was placed onto microtiter wells and the solvent was evaporated.After blocking the nonspecific binding with buffer A, the mixture ofCD14 (1 μg/ml) and phospholipid liposomes (20 μg/ml) in buffer A, whichwere preincubated at 37° C. for 1 h, were added and incubated at 37° C.for 1 h. The binding of CD14 to LPS was detected using anti-CD14monoclonal antibody. The ELISA based absorbance of CD14 bound to LPS wasdefined as 100%. The data shown are the means+S.E. from three separateexperiments with duplicate samples in each experiment. *: p<0.05, **:p<0.01, when compared with LPS-CD14 binding in the absence ofphospholipids.

FIG. 14. Monoclonal antibodies specific for the LPS binding site inhibitCD14 interaction with POPG and PI. POPG (A) or PI (B) were coated ontomicrotiter plates. After blocking the nonspecific binding with buffer A,the mixture of CD14 (1 μg/ml) and monoclonal antibodies or isotypecontrol IgG (50 μg/ml) in buffer A, which were preincubated at 37° C.for 1 h, were added and incubated at 37° C. for 1 h. The binding of CD14to phospholipids was detected using sheep anti-CD14 polyclonal antibody,and the ELISA based absorbance of CD14 bound to phospholipid was definedas 100%. The data shown are the means+S.E. from three separateexperiments with duplicate samples in each experiment. *: p<0.05, whencompared with CD14-binding in the absence of monoclonal antibody. (C)CD14 (2 μg) was coated onto microtiter plates and nonspecific bindingwas blocked with buffer A. Monoclonal antibodies or isotype control IgG(50 μg/ml) in buffer A were added and incubated at 37° C. for 1 h. TheCD14 was detected using sheep anti-CD14 polyclonal antibody, and theELISA based absorbance of solid phase CD14 alone was defined as 100%.

FIG. 15. Anionic phospholipid antagonism of LPS action does not requireLBP. (A) 10 ng/ml of LPS and different concentrations of liposomes wereadded to monolayer cultures of differentiated U937 cells in RPMI withoutserum. After 6 h of stimulation, media were collected and TNF-Aproduction was determined by ELISA. LPS stimulation without phospholipidwas defined as 100%. (B) CD14 (2 μg) was adsorbed onto microtiter wells.After blocking nonspecific binding with buffer A, the mixture of LBP (1μg/ml) and phospholipid liposomes (20 μg/ml) in buffer A, which werepreincubated at 37° C. for 1 h, was added and further incubated at 37°C. for 1 h. The binding of LBP to CD14 was detected using anti-LBPpolyclonal antibody. The ELISA based absorbance of LBP bound to CD14 wasdefined as 100%. The data shown are the means±S.E. from three separateexperiments with duplicate samples in each experiment. *: p<0.05, whencompared with CD14-LBP binding in the absence of phospholipids.

FIG. 16. MD-2 preferentially binds POPG. (A) POPG (1.25 nmole) wasplaced onto microtiter wells and the solvent evaporated. After blockingnonspecific binding with buffer A, MD-2, sTLR4 and PstB2 (1 μg/ml) inbuffer A were added and incubated at 37° C. for 1 h. The binding ofrecombinant proteins to POPG was detected using anti-His antibody. (B)Phospholipids (1.25 nmole) were placed onto microtiter wells and thesolvent evaporated. After blocking, MD-2 (1 μg/ml) in buffer A was addedand incubated at 37° C. for 1 h. The binding of MD-2 to phospholipidswas detected using anti-His antibody. The ELISA based absorbance of MD-2bound to POPG was defined as 100%. The data shown are the means+S.E.from three separate experiments each with duplicate determinations.

FIG. 17. POPG disrupts MD-2 interaction with TLR4. sTLR4 (100 ng) wasadsorbed onto microtiter wells. After blocking nonspecific binding withbuffer A, the mixture of MD-2 (1 μg/ml) and phospholipid liposomes (20μg/ml) (A) or different concentrations of phospholipids (B) in buffer A,which were preincubated at 37° C. for 1 h, was added and incubated at37° C. for 2 h. The binding of MD-2 to sTLR4 was detected usingHRP-conjugated anti-V5 monoclonal antibody. The ELISA based absorbanceof MD-2 binding without phospholipids was defined as 100%. The datashown are the means+S.E. from three separate experiments each withduplicate determinations. *: p<0.05, **: p<0.01, when compared withMD-2-sTLR4 binding in the absence of phospholipids.

FIG. 18 is a graph showing TNFα production with TLR agonists and POPGantagonism in RAW264.7 at 24 hr.

FIG. 19 is a graph showing IL-8 production with TLR agonists and POPGantagonism in Beas2B epithelial cells.

FIG. 20 is a graph showing IL-8 production in NHBE with polyIC and POPG.

FIG. 21 is a graph showing IL-8 production in human neutrophils and itsantagonism by POPG.

FIG. 22 is a graph showing that unsaturated phosphatidylglycerol (POPG)inhibits IL-6 and IL-8 production by BEAS2B and normal human bronchialepithelial (NHBE) challenged by infection with Respiratory SyncytialVirus (RSV).

FIG. 23 is a digital image showing that unsaturated phosphatidylglycerol(POPG) prevents the cytopathic effects of RSV upon BEAS2B cells.

FIG. 24 is a digital image showing that unsaturated phosphatidylglycerol(POPG) prevents the cytopathic effects of RSV upon NHBE cells.

FIG. 25 is a digital image showing that unsaturated phosphatidylglycerol(POPG) prevents viral replication in BEAS2B and NHBE cells.

FIG. 26 is a graph showing that unsaturated phosphatidylglycerol (POPG),but not unsaturated phosphatidylcholine (POPC) inhibits cytokineproduction in BEAS2B and NHBE cells challenged with RSV.

FIG. 27 is a digital image showing that unsaturated phosphatidylglycerol(POPG), but not unsaturated phosphatidylcholine (POPC) prevents thecytopathic effects of RSV upon BEAS2B cells.

FIG. 28 is a digital image showing that unsaturated phosphatidylglycerol(POPG), but not unsaturated phosphatidylcholine (POPC), prevents thecytopathic effects of RSV upon NHBE cells.

FIG. 29 shows that saturated PtdGro does not block the anti-inflammatoryeffects of SP-A upon macrophages stimulated with LPS, andunsaturated-PtdGro exerts potent anti-inflammatory effects on thesemacrophages.

FIG. 30 shows that the inhibitory effect of phosphatidylglycerols onLPS-induced inflammatory mediator production is molecular speciesspecific.

FIG. 31 shows that POPG, DMPG and PI antagonize the effects of LPS onprimary human alveolar macrophages.

FIG. 32 shows that POPG inhibits activation of RAW 264.7 macrophages bymultiple TLRs.

FIG. 33 shows that POPG inhibits activation of primary bronchialepithelial cells by multiple TLRs.

FIG. 34 shows that that POPG suppresses inflammatory cytokine productionin BEAS2B, and normal human bronchial epithelial cells, induced byRespiratory Syncytial Virus (RSV).

FIG. 35 shows that POPG prevents the killing of BEAS2B cells by RSV.

FIG. 36 shows that POPG prevents the killing of normal human bronchialepithelial cells by RSV.

FIG. 37 shows that POPG binds RSV with high affinity and specificity,and inhibits IL-8 production from epithelial cells in aconcentration-dependent manner.

FIG. 38 shows that POPG blocks the binding of RSV to epithelial cells.

FIG. 39 shows that POPG arrests the progression of RSV infection.

FIG. 40 shows the quantification of the arrest of plaque progression.

FIG. 41 shows that POPG suppresses RSV infection in vivo.

FIG. 42 shows that nanodisc POPG suppresses activation of TLR4 inmacrophages.

FIG. 43 shows that nanodisc POPG suppresses activation of TLRs 2, 3 and6 in epithelial cells.

FIG. 44 shows that nanodisc PG of various species is effective atpreventing cytopathology in cells induced by RSV.

FIG. 45 shows that nanodisc PG of various species is effective atpreventing cytopathology in cells induced by RSV.

FIG. 46 shows that nanodisc PG of various species is effective atpreventing cytopathology in cells induced by RSV.

FIG. 47 shows that both liposome and nanodisc POPG inhibit plaqueformation by RSV.

FIG. 48 shows that both liposome and nanodisc POPG inhibit plaqueformation by RSV.

FIG. 49 shows that liposome POPG prevents the cytopathology and theinflammation induced by influenza virus.

FIG. 50 shows that liposome POPG prevents the cytopathology and theinflammation induced by influenza virus.

DESCRIPTION OF THE INVENTION

The present invention generally relates to the discovery by the presentinventor that particular surfactant phospholipids, and particularly,anionic phospholipids, are potent inhibitors of inflammation.Specifically, the inventor has discovered that unsaturatedphosphatidylglycerols (PGs or PtdGro), including, but not limited topalmitoyl-oleoyl-phosphatidylglycerol (POPG), unsaturatedphosphatidylinositols (PIs or PtdIns), and selected short chainsaturated phospholipids, including, but not limited to, short chainsaturated PGs (e.g., dimyristoyl-phosphatidylglycerol (DMPG) or14:0/14:0-PtdGro), are potent inhibitors of inflammation. In addition,the present invention relates to the extension of this discovery to theuse of a class of lipids for the prevention or inhibition ofinflammation. In particular, in addition to the above-described lipids,the invention relates to the use of any anionic lipid that has thefollowing characteristics: (1) has a hydrophobic portion; (2) has anegatively charged portion; and (3) has an uncharged, polar portion, forthe prevention or inhibition of inflammation. Such lipids include, butare not limited to, the above-mentioned phospholipids, anionicsphingolipids, anionic glycerolipids (e.g., anionic diglycerides fromplants, such as SQV-diglycerides). In addition, the invention relates tothe use of compounds closely related to unsaturated PG and unsaturatedPI, and particularly lyso-PG and lyso-PI, including, but not limited to,saturated or unsaturated lyso-PG, and saturated or unsaturated lyso-PI,for the prevention and or inhibition of inflammation.

The inhibitory activity of the lipids and compounds of the invention canbe attributed to activation of the specific toll receptors, TLR1, TLR2,TLR3, TLR6, TLR7, and TLR8, as well as TLR4, and in some embodiments,TLR10. Accordingly, the present invention relates to homogeneouspreparations of these anionic lipids and related compounds, as well asvarious compositions comprising these anionic lipids and relatedcompounds, and the use of these anionic lipids and related compoundsand/or compositions thereof, for the prevention and/or treatment ofinflammation, and particularly inflammation associated with theactivation of TLR1, TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR10, andinfections, conditions and diseases related to such activation.

The present invention also relates to the use of these anionic lipidsand related compounds and/or compositions containing such anionic lipidsand related compounds, to prevent and/or treat viral infections, andmore particularly, certain respiratory infections, including, but notlimited to, respiratory syncytial virus (RSV) infection.

The present invention also relates to special formulations of pulmonarysurfactant for the enhancement of anti-inflammatory and anti-viralproperties of surfactant, which can be used in any of the methodsdescribed herein and in the treatment of any inflammatory condition ordisease and/or infection by a pathogen. These formulations are describedbelow.

Unsaturated PtdGro is a normal constituent of human pulmonarysurfactant. However, when tested as an isolated lipid preparation, theinventor demonstrates herein that PtdGro lipid suppresses inflammation,which is attributable to activation of TLR1, TLR2, TLR3, TLR4, TLR6,TLR7, and TLR8. The lipid also suppresses viral infection (e.g., RSV)due to TLR4/CD14 ligation and viral inflammation due to TLR3. Withoutbeing bound by theory, the inventor also believes that TLR10 may be atarget of this inhibitory action.

More specifically, the inventor first examined the anti-inflammatoryeffect of surfactant phospholipids upon LPS-induced inflammation inmacrophages (see Example 1). The purpose of this investigation was todetermine 1) if minor surfactant lipids can act as LPS antagonists, 2)the molecular specificity of that antagonism, and 3) the mechanism ofsurfactant lipid mediated antagonism. In particular, the inventordemonstrated that anionic surfactant lipids play an important role inregulating pulmonary inflammation in response to LPS. The inventor'sdata provides strong evidence that POPG and PI, which are minorcomponents of pulmonary surfactant, effectively inhibit LPS-inducedinflammatory responses by U937 cells, primary rat alveolar macrophagesand primary human alveolar macrophages. POPG and PI block LPS-inducedphosphorylation of MAPKs and IkBα. These anionic lipids also prevent LPSinduced degradation of IkBα, and MKP-1 expression, indicating that LPSsignaling is not transmitted from TLR4. Consistent with this latterinterpretation is the finding that POPG and PI also inhibit the bindingof BODIPY-LPS to RAW264.7 cells. These findings identify an intrinsicsystem within the lung that suppresses inflammation and protects thedelicate alveolar compartment from damage. The action of POPG and PIappears complementary to that of the pulmonary surfactant collectins,SP-A and SP-D, that also function to suppress inflammation within thelung (Wright, J. R. 2005, Nat Rev Immunol 5:58). By maintaining a basalsuppressive state within the conducting and gas exchange regions of theorgan, the lung remains largely unresponsive to low level exposure toairborne particulate matter that contains LPS. This type of suppressivestate ensures that the alveolar epithelium at the interface with theexternal environment is not chronically inflamed, as a consequence ofrepeated minor exposure to inflammatory stimuli.

The LPS antagonism of POPG and PI demonstrated herein is specific, sinceother phospholipids such as PC, PE and SM are without effect. Incomparison to POPG and PI, another anionic phospholipid, PS, is only aweak antagonist of LPS action. Moreover, even within the class of PGsthere is specificity of action. DPPG and DSPG failed to antagonize LPSaction upon macrophages, and among shorter chain saturated PGs, onlyDMPG acted as an effective antagonist. Human surfactant is highlyenriched in POPG but contains no DMPG.

The present inventor's findings identify an important role for the minoracidic lipids of pulmonary surfactant in suppressing inflammation withinthe alveolar compartment of the lung that is induced by activation ofTLR4. The site of action of PtdGro and PtdIns appears to be at the cellsurface of macrophages, and perhaps other cells where the recognition ofLPS by TLR4 is disrupted.

Example 2 provides a demonstration of some of the mechanisms by whichthe surfactant lipids act. Specifically, the inventor provides evidenceherein that two anionic pulmonary surfactant phospholipids (POPG and PI)inhibit LPS-induced inflammatory responses from macrophages. It isdemonstrated that POPG and PI bind to CD14 and form stable complexesdetectable by ELISA. The interactions between POPG and CD14 disrupt CD14binding to LPS and LBP. In addition to binding CD14, POPG also binds toMD-2. The POPG binding to MD-2 disrupts the interaction of this proteinwith TLR4. From these data, it is concluded that CD14 is a common ligandfor PI and POPG antagonism of LPS action. In addition, the antagonism ofLPS-induced inflammation by POPG is enhanced by interaction of thislipid with MD-2.

The crystal structure of mouse CD14 demonstrates the protein has LPSbinding pockets at its N-terminus (Kim et al., 2005, J Biol Chem280:11347). Four LPS binding regions have been identified within theNH₂-terminal 65 residues of CD14 (Cunningham et al., 2000, J Immunol164:3255). Monoclonal antibodies biG14 and MEM-18 bind to regionscorresponding to the third and fourth pockets, and block LPS binding.Both POPG and PI strongly bind to CD14 and the monoclonal antibodiesbiG14 and MEM-18 compete for the binding of CD14 to these lipids. Thesedata demonstrate significant overlap between the LPS and anionicsurfactant phospholipid binding sites. From single-residue mutationexperiments, charge reversal mutations within binding regions 3 and 4had the greatest effect on LPS binding (Cunningham et al., supra). Sincethe hydrophilic portion of LPS is also negatively charged, the anionicphospholipids may compete with LPS by interfering with charge dependentinteractions with CD14. Kim et al suggested the hydrophobic portion ofLPS binds to the first and second NH₂-terminal pockets since these arethe only hydrophobic surfaces large enough to accommodate acyl portionsof LPS (Kim et al., supra). POPG and DMPG bind to CD14 and antagonizethe actions of LPS. In contrast DPPG, dilauroyl-PG and dioctanoyl-PGfail to antagonize LPS action. The molecular species specificity of PGaction demonstrate that fatty acid structure is also an importantdeterminant of the interaction of phospholipids with the hydrophobicpockets in CD14.

Mueller et al. reported that LBP was a target for the inhibitoryfunction of anionic phospholipids including PG, PI and cardiolipin(Mueller et al., 2005, J Immunol 172:109). However, the presentinventors could only demonstrate that POPG attenuates the binding of LBPto CD14. Furthermore, in serum free media without LBP, anionicphospholipids still inhibit LPS-induced inflammation. Thus, the presentinventor's data show that PI and POPG can antagonize LPS action bymechanisms other than interference with LBP-LPS interactions. LPS alsobinds to MD-2 without a requirement for either LBP or CD14 (Viriyakosolet al., 1995, J Biol Chem 270:361). MD-2 binds to the extracellular TLR4domain and a complex of MD-2 and TLR4, but not TLR4 alone can interactwith LPS (Hyakushima et al., 2004, J Immunol 173:6949). POPG binds toMD-2 with high affinity. Interestingly, the interaction of POPG withMD-2 inhibits the binding of the protein to TLR4 and subsequentlyantagonizes LPS action. The interaction between POPG and MD-2 isspecific since PI fails to bind the protein. Previous protein sequenceanalysis has identified MD-2 as a protein related to fungal PG and PIbinding and transfer proteins (Inohara et al., 2002, Trends Biochem Sci27:219). According to Gioannini et al, the most efficient response toendotoxin occurs when it is sequentially transferred from LBP to CD14and finally MD-2 to engage TLR4 dependent intracellular signaling(Gioannini et al., 2004, Proc Natl Acad Sci USA 101:4186). PI interfereswith the interactions between LPS and CD14 but POPG acts at multiplesteps of protein-LPS recognition. Thus, POPG appears more broadlydirected at multiple pattern recognition proteins.

Collectively, the inventor's data described herein demonstrate that theanionic pulmonary surfactant lipids play a crucial role in suppressinginflammatory responses in the delicate alveolar compartments of thelung. Unnecessary and persistent inflammation in this region is likelyto compromise the efficiency of O₂/CO₂ exchange. The lung appearsuniquely poised between suppression and activation of inflammatoryresponses, with the basal homeostatic condition favoring suppression.This suppression depends on the lipids, PG and PI, and the pulmonarycollectins, SP-A and SP-D. The presence of multiple surfactantcomponents with these activities, provides a means of expanding therepertoire of pathogen derived pro-inflammatory components that can beantagonized during routine daily exposure. The net result appears tomaintain the lung in a quiescent state until a critical threshold isreached that finally allows inflammation to proceed. The loss of controlof inflammation can lead to septic shock syndrome, acute lung injury andacute respiratory distress syndrome (ARDS), which remain untreatablediseases (Rubenfeld et al., 2005, N Engl J Med 353:1685). The inventor'sfindings that anionic surfactant phospholipids regulate the innateimmune system and directly interact with receptors are important forunderstanding fundamental mechanisms of host defense in the lung. Thesecurrent findings cause the inventor to propose herein that exogenoussupplementation of the bronchoalveolar compartment of the lung withanionic lipids will provide a means of controlling excessiveinflammation within this organ.

The present inventor has also demonstrated that the anionic surfactantphospholipids described herein are capable of inhibiting the activity ofother TLRs than TLR4 in a specific manner. In particular, when tested asan isolated lipid preparation, PtdGro lipid suppresses inflammationattributable to activation of TLR1, TLR2, TLR3 TLR4, TLR6, TLR7 and TLR8(see Example 3). Inflammation attributable to activation of other TLRs,including TLR5, TLR9 and TLR11, are apparently not affected by thephospholipids that form the basis of this invention. TLR10 may beincluded among the list of TLRs which are inhibited by the method of theinvention, for the purposes of this disclosure.

Accordingly, methods that target inflammation associated with TLR1,TLR2, TLR3 TLR4, TLR6, TLR7, TLR8, and/or TLR10, and infections,conditions or diseases associated with these TLRs, are embodiments ofthe invention. These TLRs have been associated, for example, withvarious bacterial infections, protozoan and fungal infections, viralinfections (e.g., Cytomegalovirus infection, Herpes simplex virusinfection, measles, Varicella-zoster virus infection, HIV infection,rhinovirus infection, parainfluenza virus infection, Human parechovirusinfection, influenza type A viral infection, Papilloma virus infection),cancer (including, but not limited to, melanoma and basal cellcarcinoma), autoimmune diseases, Bowen's disease, condyloma, genitalwarts, and mollusca contagiosa.

The inventor also provides evidence herein that the particularphospholipid preparations of the invention suppress infection byrespiratory syncytial virus (RSV), e.g., due to TLR4/CD14 ligation (notassociated with LBP), and viral inflammation due to TLR3. The inventor'sresults in RSV indicate that preparations of the lipids described herein(e.g., any of the anionic lipids and related compounds, including, butnot limited to, unsaturated PGs, unsaturated PIs, certain saturatedshort chain PGs or PIs, anionic sphingolipids, anionic glycerolipids,saturated or unsaturated lyso-PG, and/or saturated or unsaturatedlyso-PI, can be used alone or in combination with other lipids oragents, and/or as a supplement to conventional surfactant preparations,to prevent and/or treat RSV infection. In addition, such preparationscan be used to prevent and/or treat other inflammatory conditions,including pulmonary infections and disorders, including in infants,children and adults, such conditions including, but not limited to,adult respiratory distress syndrome (ARDS), acute lung injury (ALI),viral infection associated with asthma, chronic obstructive pulmonarydisease (COPD), pneumonia, bronchitis, tuberculosis, reactive airwaydisease syndrome, interstitial lung disease, rhinitis, and parasiticlung disease.

Moreover, the inventor has demonstrated that surface dilution andrandomization of POPG within a single vesicle of lipids significantlydiminishes the potency of the lipid as an antagonist of LPS action.Specifically, the efficacy of PtdGro as an anti-inflammatory agent wastested after mixture with pulmonary surfactant lipids and hydrophobicproteins. Under conditions where PtdGro is randomly mixed withsurfactant lipids, such as it would be provided using most commerciallyavailable preparations of surfactant, its effectiveness was greatlyreduced. The inventor's data showed that in order to approximate theactivity of POPG alone observed in other experiments, randomized POPG(i.e., POPG randomly mixed with surfactant lipids) must constitutenearly 50% of the total lipid present in a surfactant lipid-containingvesicle. In contrast, admixture of pure POPG vesicles and randomizedsurfactant lipid vesicles had essentially no detrimental effect upon theactivity of POPG as an LPS antagonist. This result also indicated thatthe combination of pure POPG vesicles with surfactant lipid vesiclesdoes not result in significant fusion and intermixing of lipids betweenvesicle bilayers. This important result indicates that the introductionof POPG vesicles into the surfactant environment of the alveolarcompartment of the lung is expected to yield physical forms of the lipidthat are capable of potently antagonizing LPS action.

Accordingly, the inventor has shown that supplementation of humansurfactant lipids with POPG liposomes improves LPS antagonism both invivo and in vitro. The in vitro experiments demonstrate that segregatedpopulations of POPG liposomes are the most effective antagonists of LPSaction. It is not yet known whether POPG can exist in segregated domainswithin the surfactant monolayer or within the alveolar hypophase presentin the lung. However, biophysical studies provide good evidence thatDPPC can exist in distinct domains in the surfactant layer and thus itis reasonable to conclude that POPG could also be present in segregateddomains (Nag et al., 1998, Biophys J 74:2983). The in vivo studiesperformed with intra-tracheal administration of POPG strongly supportthe embodiments of the invention related to the provision ofsupplemental POPG to effectively attenuate lung inflammation in vivo,and are consistent with a model in which the lipid remains in asegregated state.

Indeed, this discovery by the inventor likely accounts for the reasonthat the presence of PtdGro in commercial surfactant preparations(including those derived from biological sources, such as porcine orbovine surfactant) has not, to the inventor's knowledge, had anydemonstrable effect as an anti-inflammatory or anti-viral agent.However, as demonstrated herein, if PtdGro is first prepared as aseparate homogeneous liposomal suspension in aqueous solution and isthen subsequently added to randomly mixed surfactant phospholipids inaqueous solution, it retains full potency as an anti-inflammatory andprobably anti-viral agent.

Therefore, it is an embodiment of the invention to provide asubstantially homogeneous preparation of the anionic lipids and/orrelated compounds of the present invention (described above and in moredetail below), which may provided for use in any of the preventative ortherapeutic methods described herein alone, or by admixture(combination, directed mixing) with other lipids, including surfactantpreparations. It is a further embodiment of the invention to providerandomly mixed surfactant preparations, wherein at least 50% of thetotal lipids in the preparation is one or more of the particular anionicphospholipids or lipids or related compounds that form the basis of thepresent invention.

Accordingly, one embodiment of the present invention relates tocompositions comprising an effective amount of at least one anioniclipid. According to the present invention, an anionic lipid useful inthe present invention has at least the following characteristics: (1)has a hydrophobic portion; (2) has a negatively charged portion; and (3)has an uncharged, polar portion. Reference to an anionic lipid useful inthe invention will be understood to refer to lipids with thesequalities. Anionic lipids useful in the invention therefore include, butare not limited to, unsaturated phosphatidylglycerol, unsaturatedphosphatidylinositol, saturated short chain phosphatidylglycerol,saturated short chain phosphatidylinositol, and derivatives of any ofsuch phospholipids (e.g., polyethylene glycol (PEG) conjugates of thesephospholipids), as well as anionic sphingolipids, anionic glycerolipids(anionic diglycerides, such as SQV-diglyceride), and any derivatives ofsuch lipids. Preferred phospholipids include, but are not limited to,unsaturated phosphatidylglycerol, unsaturated phosphatidylinositol,palmitoyl-oleoyl-phosphatidylglycerol (POPG), anddimyristoyl-phosphatidylglycerol (DMPG). In one preferred embodiment,the phospholipids are selected frompalmitoyl-oleoyl-phosphatidylglycerol (POPG) and/or phosphatidylinositol(PI) and/or derivatives thereof. The invention also includes the use ofcompounds closely related to unsaturated PG and unsaturated PI asinhibitors of inflammation, and particularly, antagonists of TLRs, andparticularly, lyso-PG and lyso-PI. Because lyso-PG and lyso-PI have muchhigher water solubility and form micellar rather than bilayerstructures, they would have greater access from the bulk solution to theTLRs. Thus, saturated or unsaturated lyso-PG, and saturated orunsaturated lyso-PI are predicted to be useful for the prevention andtreatment of inflammation according to the invention, and as antagonistsof the activation of TLRs 1,2,3,4,6,7, and 8, and in some embodiments,TLR10. General reference to “related compounds” with respect to theanionic lipids of the invention, refers to these lyso-PG and lyso-PIcompounds, or other similar compounds.

Phosphatidylglycerol (PG) is a ubiquitous phospholipid that is a majorcomponent of bacterial cell membranes and a lesser component of animaland plant cell membranes. In animal cells, PG may serve primarily as aprecursor for diphosphatidylglycerol (cardiolipin). PG is the secondmost abundant phospholipid in lung surfactant in most animal species. Aparticularly useful PG in the present invention ispalmitoyl-oleoyl-phosphatidylglycerol (POPG).

Phosphatidylinositol (PI) is a key membrane constituent and is aparticipant in essential metabolic processes in all plants and animals(and in some bacteria (Actinomycetes)), both directly and via a numberof metabolites. It is an acidic (anionic) phospholipid that in essenceconsists of a phosphatidic acid backbone, linked via the phosphate groupto inositol (hexahydroxycyclohexane). In most organisms, thestereochemical form of the last is myo-D-inositol (with one axialhydroxyl in position 2 with the remainder equatorial), although otherforms (scyllo- and chiro-) have been found on occasion in plants. PI isformed biosynthetically from the precursor CDP-diacylglycerol byreaction with inositol, catalysed by the enzyme CDP-diacylglycerolinositol phosphatidyltransferase.

Unsaturated PGs and PIs are defined herein as any PG or PI with one ormore double bonds in the fatty acid chain.

Saturated PGs or PIs are defined herein as any PG or PI without a doublebond (i.e., the chains are fully saturated with hydrogens). A saturatedshort chain PG or PI useful in the present invention includes anysaturated 14 carbon or shorter PG or PI with anti-inflammatoryproperties as described herein. A particularly preferred saturated shortchain PG includes, but is not limited to,dimyristoyl-phosphatidylglycerol (DMPG).

According to the present invention, an compositions containing an“effective amount” of an anionic lipid or related compound of theinvention contain an amount of the specific anionic lipid or relatedcompound effective to inhibit an inflammatory process in vitro or invivo, or to inhibit viral infection in vitro or in vivo, as measured byany suitable technique for measuring such activity, several of which aredescribed herein. Effective amounts of anionic lipids or relatedcompounds of the invention to be included in a composition are describedin more detail below.

In one aspect of the invention, the anionic lipids or related compoundsare provided in a homogeneous lipid preparation comprising, consistingessentially of, or consisting of one or more of the anionic lipids orrelated compounds described above, and/or derivatives of any of suchanionic lipids or related compounds. In one embodiment, any of theabove-described lipid preparations further comprise any other lipid orlipid derivative that is useful in a surfactant preparation, useful in atherapeutic preparation, and/or useful for stabilizing the bilayer oflipids in a lipid preparation and/or decreasing leakage of encapsulatedmaterial. In one embodiment, any of the above-described lipidpreparation further comprise antioxidants, which are useful forinhibiting oxidation of the lipids in lipid preparation.

According to the invention, a lipid preparation useful in the inventioncan include any stabilized form of lipid that would be useful in amethod of the invention, and particularly, any lipid that is stabilizedby protein or another suitable compound. For example, lipid preparationsuseful in the invention include, but are not limited to, liposomes, andprotein-stabilized lipid forms (e.g., non-liposomal lipids stabilized bythe use of a lipoprotein, e.g., see Nanodisc™, Nanodisc, Inc.).

According to the present invention, a liposome (also referred to as aliposomal preparation or liposomal composition) is a spherical,microscopic artificial membrane vesicle consisting of an aqueous coreenclosed in one or more phospholipid layers. Liposomes can also begenerally defined as self closed spherical particles with one or severallipid membranes. Liposomes can be composed of naturally-derivedphospholipids with mixed fatty acid chains or prepared from syntheticlipids with well-defined lipid chains. Depending on the number of themembranes and size of the vesicles, liposomes are considered to be largemultilamellar vesicles (LMV) with sizes up to 500 nm, small unilamellarvesicles (SUV) with sizes <100 nm, and large unilamellar vesicles (LUV)with sizes >100 nm. Liposomes and liposome preparation methods are wellknown in the art, and several example of liposomes useful in the presentinvention, as well as methods of producing such liposomes andcompositions comprising such liposomes, is described in the Examples. Astabilized lipid, such as a protein- or lipoprotein-stabilized lipid,can be prepared using any method known in the art.

In one exemplary embodiment, the lipid in the lipid preparation iscomposed of pure unsaturated PG, pure unsaturated PI, pure saturatedshort chain PG, pure saturated short chain PI, pure anionicsphingolipid, pure anionic glycerolipid, pure unsaturated lyso-PG, puresaturated lyso-PG, pure unsaturated lyso-PI, pure saturated lyso-PI, orany combinations thereof. In one exemplary embodiment, the lipid in thelipid preparation is composed of purepalmitoyl-oleoyl-phosphatidylglycerol (POPG),dimyristoyl-phosphatidylglycerol (DMPG), pure unsaturated PI, pureunsaturated PG, or any combinations thereof. Similarly, lipidpreparations can be composed of any of these anionic lipids or relatedcompounds, in combination with one or more different phospholipidsand/or other lipid(s) and/or related compounds.

Preferred compositions for use in the invention provide an amount of theanionic lipids or related compounds described as useful in the presentinvention to provide a therapeutic or anti-inflammatory or anti-pathogen(e.g., anti-viral) effect when administered to an individual. Forexample, as discussed above, prior to the present invention, thepresence of effective anionic phospholipids of the invention, such asPtdGro, in commercial surfactant preparations (including those derivedfrom biological sources, such as porcine or bovine surfactant) has not,to the inventor's knowledge, had any demonstrable effect as ananti-inflammatory or anti-viral agent. This is because surface dilutionand randomization of the effective phospholipids of the invention withina single vesicle of lipids significantly diminishes the potency of thelipid, as demonstrated in the Examples. As the Examples illustrate, inorder to approximate the activity of the anionic phospholipid alone,randomized anionic phospholipid of the invention (e.g., POPG randomlymixed with surfactant lipids) must constitute nearly 50% of the totallipid present in a surfactant lipid-containing vesicle. However, ifprovided as a separate, homogeneous lipid preparation, even when admixedwith other lipids after production of the lipid preparation, the anionicphospholipid of the invention can be provided in smaller quantities.This element of the invention can be extended to the other anioniclipids or related compounds of the invention.

Therefore, several different compositions of the anionic lipids orrelated compounds described herein are envisioned for use in theinvention. In one embodiment, the invention provides a homogeneous lipidpreparation consisting of the anionic lipid or related compound. As usedherein, a “homogeneous” lipid preparation consisting of a specifiedanionic lipid or related compound or combination of specified anioniclipids or related compounds, means that the lipid preparation (e.g., thelipid vesicles or smaller portions) contains only the specified anioniclipid or related compound or a combination of specified anionic lipidsor related compounds (e.g., a pure preparation of the specifiedphospholipid(s)), and is substantially or completely free of otherphospholipids or other lipids. A homogeneous preparation of a specifiedanionic lipid or related compound can contain other non-lipid agents, ifdesired, such as antioxidants, a targeting moiety (described below), oranother therapeutic agent (e.g., a protein, and antibody, a smallmolecule or drug). A homogeneous lipid preparation of the invention canbe provided alone or with a pharmaceutically acceptable carrier,including an excipient or buffer, or in a composition with other agentsor lipid preparations.

It is one embodiment of the invention to administer a homogeneous lipidpreparation of an anionic lipid or related compound of the invention inthe absence of any other lipids, although in other embodiments, theadditive effects of other lipids, such as other lipids contained insurfactant, may be desirable and useful. In these alternate embodiments,the invention provides compositions that allow for the provision of suchadditional lipids and/or combinations of lipids, without losing theeffectiveness of the particular anionic lipids or related compoundsdescribed herein. Such compositions are described below. Accordingly, inone aspect of the invention, the composition comprises other lipidpreparations.

In one embodiment, the invention provides a composition comprising ahomogeneous lipid preparation of the anionic lipid(s) or relatedcompound(s) of the invention and at least one additional agent. Theadditional agent can include any pharmaceutical carrier, as discussedabove, or an additional agent for the treatment of inflammation orpathogen infection (e.g., an anti-viral agent), for example.

Suitable anti-inflammatory agents include, but are not limited to,cytokine inhibitors, chemokine inhibitors, chemoattractant inhibitors,Cox inhibitors, leukotiene receptor antagonists, leukotriene synthesisinhibitors, inhibitors of the p38 MAP kinase pathway, glucocorticoids.More specifically, anti-inflammatory compounds can include, but are notlimited to: any inhibitor of eicosanoid synthesis and release, includingany Cox-2 inhibitor; Cox-1 inhibitors; inhibitors of some certainprostaglandins (prostaglandin E(2); PGD(2)), inhibitors of certainleukotrienes (LTB₄); classes of antibiotics with known direct orindirect anti-inflammatory effects, including macrolides (e.g.azithromycin) and fluoroquinolones (e.g., levofloxacin; moxifloxacin;gatifloxacin); inhibitors of p38 MAP kinase; inhibitors of the functionof pro-inflammatory cytokines and chemokines, including antagonists oftumor necrosis factor (TNF), antagonists of interleukin-8 (IL-8);transforming growth factor beta (TGF-beta), β-agonists (long or shortacting), antihistamines, phosphodiesterase inhibitors, corticosteroids,and other agents.

According to the present invention, a “pharmaceutically acceptablecarrier” includes pharmaceutically acceptable excipients and/orpharmaceutically acceptable delivery vehicles, which are suitable foruse in the administration of a preparation, formulation or composition,including a liposomal composition or preparation, to a suitable in vivosite. A suitable in vivo site is preferably any site whereininflammation or infection by a pathogen, for example, is occurring or isexpected to occur. Preferred pharmaceutically acceptable carriers arecapable of maintaining a formulation of the invention in a form that,upon arrival of the formulation at the target site in a patient (e.g.,the lung tissue), the formulation is capable of acting at the site,preferably resulting in a beneficial or therapeutic benefit to thepatient. A delivery vehicle for a protein or agent can include the lipidpreparation itself, if another agent is included, although in mostembodiments of the invention, the lipid preparation is also atherapeutic agent as described herein (e.g., the lipid preparation canserve one or both functions).

Suitable excipients of the present invention include excipients orformularies that transport or help transport, but do not specificallytarget, a composition or formulation to a cell or tissue (also referredto herein as non-targeting carriers). Examples of pharmaceuticallyacceptable excipients include, but are not limited to water, phosphatebuffered saline, Ringer's solution, dextrose solution, serum-containingsolutions, Hank's solution, other aqueous physiologically balancedsolutions, oils, esters and glycols. Aqueous carriers can containsuitable auxiliary substances required to approximate the physiologicalconditions of the recipient, for example, by enhancing chemicalstability and isotonicity. Suitable auxiliary substances include, forexample, sodium acetate, sodium chloride, sodium lactate, potassiumchloride, calcium chloride, and other substances used to producephosphate buffer, Tris buffer, and bicarbonate buffer. Auxiliarysubstances can also include preservatives, such as thimerosal, m- oro-cresol, formalin and benzol alcohol. Formulations of the presentinvention can be sterilized by conventional methods and/or lyophilized.

A lipid preparation useful in the present invention can be modified totarget to a particular site in a patient, thereby targeting and makinguse of the anionic lipids or related compounds and any agents carried bythe lipid preparation at that site. Suitable modifications includemanipulating the chemical formula of the lipid preparation and/orintroducing into the lipid preparation a targeting agent capable ofspecifically targeting the lipid preparation to a preferred site, forexample, a preferred cell type. Suitable targeting agents includeligands capable of selectively (i.e., specifically) binding anothermolecule at a particular site. Examples of such ligands includeantibodies, antigens, receptors and receptor ligands.

In one embodiment, a particularly preferred composition suitable for usein the invention comprises a preparation (e.g., a lipid preparation) ofrandomly mixed anionic lipids or related compounds (any combination),and preferably, randomly mixed surfactant phospholipids or lipids (e.g.,any combination of lipids found in surfactant), combined with (added to,mixed gently with, in admixture with) a homogeneous lipid preparation ofthe anionic lipids or related compounds useful in the present invention.In this embodiment, the combining of the randomly mixed lipids with thehomogeneous lipid preparation of the anionic lipids or related compoundsis performed in a manner that does not result in significant fusionand/or intermixing of lipids between the vesicle bilayers (e.g., betweenthe randomly mixed lipid preparations and the pure or homogeneous lipidpreparation of anionic lipids or related compounds. By producing ahomogeneous preparation of the desired anionic lipids or relatedcompounds and then adding it to another preparation of lipids, such as arandomized surfactant preparation, the inventor has discovered that thebiological activity of the anionic lipids or related compounds describedherein (e.g., anti-inflammatory, anti-pathogen, including anti-viral) ismaintained. In this embodiment of the invention, it is preferred thatthe homogeneous lipid preparations of the anionic lipids or relatedcompounds of the invention comprise at least 1% of the total lipids inthe composition (e.g., the total lipids being those present in thehomogeneous preparation and the added randomly mixed surfactantpreparation), or at least 5%, or at least 10%, or at least 15%, or atleast 20%, or at least 25%, or at least 30%, or at least 35%, or atleast 40%, or at least 45%, or at least 50%, of the total lipids in thecomposition.

In another embodiment of the invention, a preparation of randomly mixedlipids is provided, and preferably a preparation of randomly mixedsurfactant lipids and phospholipids, wherein the preparation containsone or more anionic lipids or related compounds useful in the presentinvention as described above. In this embodiment, the anionic lipid(s)or related compounds comprises at least about 30% of the total lipids inthe randomly mixed surfactant lipids, or at least about 35%, or at leastabout 40%, or at least about 45%, or at least about 50%, or at leastabout 55%, or at least about 60%, or at least about 65%, or at leastabout 70%, or at least about 75%, or at least about 80%, or at leastabout 85%, or at least about 90%, or at least about 95%, of the totallipids in the randomly mixed surfactant lipids (or any amount between atleast 30% and 100%, in whole number increments, e.g., 30%, 31%, 32%,etc.).

Preparations of randomly mixed lipids, and particularly, randomly mixedsurfactant lipids can be made using techniques known in the art and arealso available commercially (e.g., see Exosurf® (Wellcome, USA, anartificial surfactant preparation); Alveofact® (Thomae, Germany,prepared from bovine BAL); Curosurf® (Chiesi, Italy, prepared fromminced porcine or bovine lung tissue) or Survanta® (Abbott, USA,prepared from minced porcine or bovine lung tissue)). Lung surfactant isa complex mixture of various phospholipids, neutral lipids andapoproteins (Doles, Ann Rev Med 1989; 40: 431-446; Jobe, N Engl J Med1993; 328: 861-868; Tegtmeyer et al., Eur Respir J, 1996, 9, 752-757).Surfactant replacement therapy has proven to be beneficial for thetreatment of the neonatal respiratory distress syndrome (Jobe, supra),and is also considered as a therapeutic option for term infants andadults with acute respiratory failure (Lewis and Jobe, Am Rev Respir Dis1993; 147:216-233). Accordingly, surfactant lipid preparations arewidely available and well known to those of skill in the art. It isbelieved that the addition of the homogeneous lipid preparations ofanionic lipids and related compounds described herein to suchpreparations will significantly enhance the use of such commercialpreparations or other surfactant preparations in the prevention andtreatment of a variety of conditions, including those described directlyabove.

The total concentration of lipids to be delivered to an individual(e.g., to the lung) according to the present invention can range fromabout 5 μmol to about 1 mmol, including any amount between, inincrements of 1 μmol. In one aspect, the amount delivered is from about40 μmol to about 800 μM, although one of skill in the art can readilydetermine the appropriate amount to be delivered. By way of example, inone embodiment, the lipid preparation comprising a given anionic lipid(e.g., unsaturated PG) is delivered in an amount suitable to replace allresident lung PG). The estimated amount of unsaturated PG in the lung isapproximately 400 umole in the entire adult lung residing in thealveolar compartment exclusive of the tissue. If the lipid preparationis to replace all resident lung PG, then 40 umol/ml×10 ml would besufficient. It is an embodiment of the invention to provide the anioniclipid(s) and/or related compound(s) to the lung in an amount deliveredthat is equivalent to between about 10% of the total resident amount ofthe same or similar lipid, to about 200% of the total resident amount.Accordingly, from a lipid preparation that is 40 umol of the lipid orcompound of the invention per ml of lipid preparation, the individualwould receive between about 1 ml and 20 ml delivered in an aqueoussuspension down the trachea, for delivery to the lungs.

In one embodiment, the lipid preparation useful in the present inventionis complexed with another agent, such as a protein or a small molecule(drug), wherein the other agent is also useful for inhibiting orpreventing inflammation or infection by a pathogen (e.g., a virus) in anindividual. Methods of encapsulating or complexing proteins and otheragents with lipids such as liposomes and protein-stabilized lipids areknown in the art. The encapsulation efficiency of proteins by lipidpreparations generally depends on interaction between the protein andthe lipid bilayer or micelle. The protein entrapment can be increased bymanipulation of the lipid preparation, or by increasing the lipidconcentration, in order to favor electrostatic interactions, whilemonitoring the ionic strength of the protein solution (Colletier et al.,BMC Biotechnology 2002, 2:9). Preferably, the amount of a proteincomplexed with lipid preparations will range from about 0.001 mg ofprotein per 1 ml lipid preparation to about 5 mg of protein per 1 mllipid preparation.

Another embodiment of the invention relates to a method to produce asurfactant composition. The method includes (a) providing a homogeneouslipid preparation of an anionic lipid(s) and/or related compound(s) asdescribed herein (e.g., an unsaturated phosphatidylglycerol, anunsaturated phosphatidylinositol, a saturated short chainphosphatidylglycerol, a saturated short chain phosphatidylinositol,anionic sphingolipid, anionic glycerolipid, unsaturated lyso-PG,saturated lyso-PG, unsaturated lyso-PI, saturated lyso-PI, or aderivative or combination thereof) and (b) adding the preparation of (a)to a preparation of randomly mixed surfactant lipids. The preparation ofrandomly mixed surfactant lipids can be produced by any suitable methodknown in the art or obtained commercially, as discussed above.Preferably, the preparation of (a) and/or (b) are in aqueous solution.Most preferably, the preparation is gently mixed to avoid significantfusion or intermixing of lipids between vesicle bilayers in (a) and (b),also as discussed above. In one aspect, the lipids in the preparation of(a) comprise at least 1% of the total lipids in the composition, or anyamount from at least 1% to at least 50% or greater, in 1% increments.

One embodiment of the present invention relates to the use of any of theanionic lipid or related compound formulations described herein,including combinations thereof, to treat or prevent inflammation or apathogen infection, and particularly a viral infection (e.g., RSV). Thepreventative and/or therapeutic methods of the invention generallyinclude the administration to an individual (any individual, includinginfants, children and adults), any one or more preparations of theanionic lipids and/or related compounds described herein, alone or incombination with other lipids or agents, and/or as a supplement toconventional surfactant preparations or other therapies.

In one embodiment, the methods of the invention are useful forpreventing or inhibiting inflammation or a pathogen infection associatedwith particular toll-like receptors, and specifically, TLR1, TLR2, TLR3,TLR4, TLR6, TLR7, TLR8, and/or TLR10. These TLRs have been associated,for example, with various bacterial infections, protozoan and fungalinfections, viral infections e.g., Cytomegalovirus infection, Herpessimplex virus infection, measles, Varicella-zoster virus infection, HIVinfection, rhinovirus infection, parainfluenza virus infection, Humanparechovirus infection, influenza type A viral infection, Papillomavirus infection), cancer (including, but not limited to, melanoma), andautoimmune diseases. Accordingly, it is an embodiment of the inventionto treat or inhibit inflammation associated with any of these conditionsor to prevent or inhibit infection by a pathogen associated with any ofthese conditions.

One particular embodiment of the invention relates to a method toprevent or inhibit (suppress, reduce) infection by respiratory syncytialvirus (RSV), as well as viral inflammation or infection by otherviruses. The method includes the administration of any of the anioniclipid and/or related compound formulations described herein, includingcombinations thereof, to an individual who has or who is at risk ofbeing infected by a virus, and particularly a virus associated with anyof the TLRs discussed above, and more particularly, with RSV. Withregard to RSV, the preparation can be administered to newborn infants,including to any newborn infant, regardless of whether the viralinfection has been detected in the infant (i.e., the invention is usefulas a prophylactic and as a therapeutic approach). Preparations of theanionic lipids and related compounds described herein can be used aloneor in combination with other lipids or agents, and/or as a supplement toconventional surfactant preparations, to prevent and/or treat RSVinfection.

The method of the invention is also useful for the prevention and/ortreatment of other pulmonary infections and disorders, including ininfants, children and adults, including, but not limited to, adultrespiratory distress syndrome (ARDS), acute lung injury (ALI), viralinfection associated with asthma, chronic obstructive pulmonary disease(COPD), pneumonia, bronchitis, tuberculosis, reactive airway diseasesyndrome, interstitial lung disease, rhinitis, and parasitic lungdisease.

In accordance with the present invention, determination of acceptableprotocols to administer a composition or formulation, including theroute of administration and the effective amount of a composition orformulation to be administered to an individual, can be accomplished bythose skilled in the art. An agent of the present invention can beadministered in vivo or ex vivo. Suitable in vivo routes ofadministration can include, but are not limited to, oral, nasal,inhaled, topical, intratracheal, transdermal, rectal, intestinal,intra-luminal, and parenteral routes. Preferred parenteral routes caninclude, but are not limited to, subcutaneous, intradermal, intravenous,intramuscular, intraarterial, intrathecal and intraperitoneal routes.Preferred topical routes include inhalation by aerosol (i.e., spraying)or topical surface administration to the skin of an animal. Preferably,an agent is administered by nasal, inhaled, intratracheal, topical, orsystemic routes (e.g., intraperitoneal, intravenous). Ex vivo refers toperforming part of the administration step outside of the patient.Preferred routes of administration for antibodies include parenteralroutes and aerosol/nasal/inhaled routes.

Intravenous, intraperitoneal, and intramuscular administrations can beperformed using methods standard in the art. Aerosol (inhalation)delivery can be performed using methods standard in the art (see, forexample, Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281,1992, which is incorporated herein by reference in its entirety).Carriers suitable for aerosol delivery are described above. Devices fordelivery of aerosolized formulations include, but are not limited to,pressurized metered dose inhalers (MDI), dry powder inhalers (DPI), andmetered solution devices (MSI), and include devices that are nebulizersand inhalers. Oral delivery can be performed by complexing a therapeuticcomposition of the present invention to a carrier capable ofwithstanding degradation by digestive enzymes in the gut of anindividual. Examples of such carriers, include plastic capsules ortablets, such as those known in the art. Administration of a compositionlocally within the area of a target cell refers to injecting thecomposition centimeters and preferably, millimeters from the target cellor tissue.

In humans, it known in the art that, using conventional methods foraerosol delivery, only about 10% of the delivered solution typicallyenters the deep airways, even using an inhaler. If the aerosolizeddelivery is by direct inhalation, one may assume a dosage of about 10%of that administered by nebulization methods. Finally, one of skill inthe art will readily be capable of converting an animal dosage to ahuman dosage using alometric scaling. For example, essentially, a scaleof dosage from mouse to human is based on the clearance ratio of acompound and the body surface of the mouse. The conversion for mg/kg is1/12th of the “no observed adverse event level” (NOEL) to obtain theconcentration for human dosage. This calculation assumes that theelimination between mouse and human is the same.

Preferred amounts of lipid preparations to be delivered to an individualhave been discussed in detail above.

In one embodiment, an effective amount of a preparation of the inventionto administer to an individual is an amount that measurably inhibits (orprevents) inflammation or infection by a pathogen in the individual ascompared to in the absence of administration of the formulation. Asuitable single dose of a formulation to administer to an individual isa dose that is capable of reducing or preventing at least one symptom,type of injury, or resulting damage, from inflammation or pathogeninfection in an individual when administered one or more times over asuitable time period. Preferably, a dose is not toxic to the individual.

One of skill in the art will be able to determine that the number ofdoses of a preparation to be administered to an individual is dependentupon the extent of the inflammatory condition or infection by a pathogenand/or the anticipated or observed physiological damage associated withsuch inflammation or infection, as well as the response of an individualpatient to the treatment. The clinician will be able to determine theappropriate timing for delivery of the formulation in a manner effectiveto reduce the symptom(s) associated with inflammation or pathogeninfection in the individual. Preferably, the agent is delivered within48 hours, and more preferably 36 hours, and more preferably 24 hours,and more preferably within 12 hours, and more preferably within 6 hours,5 hours, 4 hours, 3 hours, 2 hours, or 1 hour, or even minutes after therecognition of a condition to be treated by a formulation of theinvention; after an event that causes inflammation in an individual orinfection of an individual, or that is predicted to cause inflammationin or infection of an individual, which can include administration priorto the development of any symptoms of inflammation or infection in theindividual.

Methods and uses directed to therapeutic compositions of the inventionare primarily intended for use in the prevention and/or treatment of adisease or condition. The term “protecting” can be generically used toconvey prevention and/or treatment. A therapeutic composition of thepresent invention, when administered to an individual, can: prevent adisease from occurring; cure the disease; delay the onset of thedisease; and/or alleviate (reduce, delay, diminish) disease symptoms,signs or causes (e.g., reduce one or more symptoms of the disease;reduce the occurrence of the disease; increase survival of theindividual that has or develops the disease; and/or reduce the severityof the disease). As such, the invention includes both preventing diseaseoccurrence (prophylactic treatment) and treating an animal that has adisease or that is experiencing symptoms of a disease (therapeutictreatment).

According to the present invention, the methods of the present inventionare suitable for use in an individual that is a member of the Vertebrateclass, Mammalia, including, without limitation, primates, livestock anddomestic pets (e.g., a companion animal). Most typically, an individualwill be a human individual. The term “individual” can be interchangedwith the term “subject” or “patient” and refers to the subject of amethod according to the invention. Accordingly, an individual caninclude a healthy, normal (non-diseased) individual, but is mosttypically an individual who has or is at risk of developing aninflammatory condition or an infection, including a viral infection, ora symptom or indicator thereof as described herein.

The following experimental results are provided for purposes ofillustration and are not intended to limit the scope of the invention.

EXAMPLES Example 1

The following experimental results demonstrate thatpalmitoyl-oleoyl-phosphatidylglycerol (POPG) and phosphatidylinositol(PI), which are minor components of pulmonary surfactant, regulated theinflammatory response of alveolar macrophages. These results show thatPOPG and PI significantly inhibited LPS-induced nitric oxide and tumornecrosis factor (TNF)-α production from rat and human alveolarmacrophages and a U937 cell line. POPG and PI reduced LPS-elicitedphosphorylation of p38MAPK, ERK, and IkB-alpha; and expression ofmitogen-activated protein kinase phosphatase (MKP-1). POPG was alsoeffective at attenuating inflammation when administered intratrachealyto mice challenged with LPS. Examination of cell surface binding byBODIPY-LPS revealed that POPG and PI inhibit LPS binding to the cellsurface in a lipid structure specific manner. These data clearlyidentify important anti-inflammatory properties of surfactantphospholipids at the environmental interface of the lung.

Experimental Procedures

Cells and Reagents. LPS (0111:B4) purified from Escherichia coli waspurchased from Sigma-Aldrich (St. Louis, Mo.). BODIPY-LPS (055:B5)purified from E. coli was purchased from InVitrogen, (Carlsbad, Calif.).PC, PG, sphingomyelin (SM), phosphatidylethanolamine (PE),phosphatidylserine (PS) and PI were purchased from Avanti Polar Lipids(Alabaster, Ala.). TNFα was from Genzyme (Cambridge, Mass.). Rabbitpolyclonal anti-p46 JNK, rabbit polyclonal anti-p38, mouse monoclonalphospho-specific p46-p54 JNK antibodies, and rabbit polyclonalanti-mitogen-activated protein kinase phosphatase (MKP)-1 antibodieswere purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).Rabbit polyclonal phospho-specific p42 ERK, rabbit polyclonal anti-p42ERK, phospho-specific p38MAPK, rabbit polyclonal anti-IkBα, andphospho-specific IkBα antibodies were obtained from Cell SignalingTechnology (Beverly, Mass.). [³H]-Leucine was from Perkin Elmer LifeSciences (Boston, Mass.). The macrophage-like cell line U937(CRL-1593.2) was obtained from American Type Culture Collection(Manassas, Va.). The cells were maintained in endotoxin-free RoswellPark Memorial Institute (RPMI) 1640 medium from Cambrex (EastRutherford, N.J.) with 10% heat-inactivated bovine growth serum (BGS;Hyclone, Logan, Utah). RAW 264.7 cells were maintained in DMEM with 10%BGS.

Isolation of Rat Alveolar Macrophages. Rat alveolar macrophages wereisolated from bronchoalveolar lavage fluid (BALF) of Sprague-Dawleyrats. The lungs were lavaged with pyrogen-free saline, and alveolarmacrophages were sedimented by centrifugation at 150×g×5 min. Isolatedmacrophages were plated at 5×10⁵ cells/well in 24-well plates (Falcon)in RPMI 1640 medium containing 10% BGS. The cells were allowed to adherefor 2 h and then used for the experiments after washing withphosphate-buffered-saline (PBS) to remove the unattached cells.

Isolation of Human Alveolar Macrophages. Human alveolar macrophages wereisolated from BALF of healthy volunteers using protocols reviewed andapproved by the National Jewish Medical Research Center IRB and theUniversity of Colorado General Clinical Research Center. The lungs werelavaged with pyrogen-free saline, and alveolar macrophages weresedimented by centrifugation at 150×g×5 min. Isolated macrophages wereplated at 5×10⁴ cells/well in 96-well plates (Falcon) in RPMI 1640medium containing 10% BGS. The cells were allowed to adhere for 48 h andthen used for the experiments after washing with PBS to remove theunattached cells.

Induction of TNF-α Secretion. U937 cells were induced to differentiateby treatment with 10 nM phorbol myristate acetate (PMA) for 48 h. Thecells (1.3×10⁵/well) were placed in 96-well plates and further incubatedin the absence of PMA for 24 h in RPMI 1640 medium containing 10% BGS.Rat alveolar macrophages (5×10⁵/well) were incubated in 24-well platesfor 2 h after isolation. The indicated concentration of phospholipidswas added to the cultures 30 min before adding LPS. After LPS additioncultures were incubated for 6 h at 37° C. at an atmosphere of 95% airand 5% CO₂. At the end of the incubation period the medium was collectedand assayed for TNF-α concentrations using an ELISA kit.

Preparation of Surfactant Lipids. Surfactant lipids were isolated fromthe bronchoalveolar lavage of Sprague-Dawley rats, 28 days afterintratracheal instillation of 25 mg of silica (˜125 mg/kg). Initially,the surfactant was purified by the method of Hawgood et. al. (25) usingNaBr density gradient centrifugation. The purified surfactant wasextracted with butanol (26) and segregated into butanol-soluble and-insoluble material. The butanol-soluble surfactant lipids wererecovered by drying under vacuum and resuspending in chloroform. Thephospholipid content was determined by the method of Rouser et al (27),and the mixture was stored at −20° C. Prior to use, an aliquot ofsurfactant lipids was initially dried under nitrogen, and subsequentlyhydrated in 20 mM Tris (pH 7.4), 150 mM NaCl buffer at 37° C. for 1 h.Finally the surfactant lipids were probe-sonicated in 5-30 s bursts with1 min cooling between bursts, to make a vesicle preparation for use inexperiments.

Analysis of Nitric Oxide Accumulation. Nitric oxide (NO) accumulation inthe supernatant was determined as previously reported (28). Briefly, ratalveolar macrophages were stimulated with LPS (10 ng/ml) or LPS plusphospholipids for 24 h. Culture supernatants (usually 100 μl) werecombined with an equal volume of Greiss reagent, and the samples wereincubated at room temperature for 10 min before the absorbance wasquantified at 550 nm. With the use of a standard curve, the nmol of NOproduced were determined and normalized to total cell number in eachsample.

Analysis of Cytokine Production. Human and mouse TNF-α ELISA kits werepurchased from BioSource (Camarillo, Calif.). Mouse KC and MIP-2Quantikine kits were purchased from R&D System. Measurements of thesecytokines were according to the manufacturers' protocols.

Measurement of MARK, IkBα and MKP-1. Monolayers of unstimulated orstimulated macrophages were lysed on ice with 250 μl of ice-cold lysisbuffer [50 mM Tris·HC1, pH 8.0, containing 137 mM NaCl, 10% (vol/vol)glycerol, 1% (vol/vol) Nonidet P-40, 1 mM NaF, 10 μg/ml leupeptin, 10μg/ml aprotinin, 2 mM Na₃VO₄, and 1 mM phenylmethylsulfonyl fluoride(29). Insoluble nuclear material was pelleted by centrifugation at14,000×g for 10 min at 4° C. and the supernatants were collected. 15 μgof protein from lysates was separated by SDS-PAGE and transferred ontonitrocellulose membranes (30). The blots were then washed inTris-Tween-buffered saline [TTBS, 20 mM Tris-HCl buffer, pH 7.6,containing 137 mM NaCl and 0.05% (vol/vol) Tween 20], blocked with 5%(wt/vol) nonfat dry milk for 1 hour, and probed according to the methoddescribed by Towbin et. al. (30) with phospho-specific antibodies top46-p54 JNK, p42/p44 ERK, and p38MAPK, or IkBα or with a polyclonalMKP-1 or IkBα antibodies in 5% (wt/vol) BSA dissolved in TTBS. With theuse of horseradish peroxidase-conjugated secondary anti-rabbit oranti-mouse antibody, bound antibodies were detected by enhancedchemiluminescence (ECL plus, Amersham Biosciences, Piscataway, N.J.). Todetermine loading of proteins between samples, the membranes were probedwith rabbit polyclonal p46 JNK, p42/p44 ERK, and p38MAPK antibodies.

Administration of LPS and Phospholipids In Vivo. Female BALB/c mice from6 to 8 weeks of age were obtained from Jackson Laboratories (Bar Harbor,Me.). Experiments were conducted under a protocol approved by theInstitutional Animal Care and Use Committee of the National JewishMedical and Research Center. Liposomes were formed using a Liposofast™(Avestin; Ottawa, Canada), which makes unilamellar liposomes of 100 nmof diameter, and then mixed with an aqueous solution containing LPS. Themixture of LPS and phospholipids was sprayed into murine trachea using aMicroSprayer™ aerosolizer (PennCentury, Philadelphia, Pa.) underisoflurene anesthesia. Delivery by MicroSprayer™ has been shown toresult in lung deposition fractions of more than 93% in primates (31).After stimulation, lungs were lavaged via the trachea with 1 ml ofHank's balanced salt solution (Invitrogen Corporation, Carlsbad,Calif.). The volume of collected BALF was measured in each sample andthe number of leukocytes was counted (Coulter Counter; CoulterCorporation, Hialeah, Fla.). Differential cell counts were determinedfrom at least 300 cells on cytocentrifuged preparations (Cytospin;Shandon Ltd., Runcorn, Cheshire, UK). Slides were stained with modifiedWright-Giemsa (Hema; Protocol, Swedesboro, N.J.) and the cellpopulations differentiated by standard hematologic procedures. Cytokinelevels in the BALF or in the supernatants of cultured airway macrophageswere measured using ELISA kits.

Binding of Phospholipids to RAW264.7 Macrophages. RAW264.7 cells (10⁶)were incubated with BODIPY-LPS (1 μg/ml) either with or withoutphospholipid liposomes (200 μg/ml) at 4° C. for 4 h. When liposomes wereadded, a 1 h preincubation preceded the addition of fluorescent LPS.After the cells were washed with PBS by centrifugation, cell adherentfluorescence was determined using FACScan. Macrophages were counted for20,000 cells and the graph was made by CellQuest software.

Statistical Analysis. All results were expressed as mean±S.E. ANOVA wasused to determine the levels of difference between all groups. Groupswere compared by unpaired two-tailed t-test. The p-value forsignificance was set at 0.05.

Results

POPG and PI Inhibit LPS-induced Production of Proinflammatory Cytokines.

In the initial studies, the inventors investigated the ability ofpurified lipids normally present as minor components of pulmonarysurfactant to modulate LPS-induced cytokine secretion. Macrophages werestimulated with LPS in the presence or absence of purified phospholipids(FIG. 1). Culture supernatants were collected and TNF-α production byU937 cells and NO production by rat alveolar macrophages was determinedPOPG and PI significantly attenuated TNF-α and NO production in aconcentration dependent manner with the maximal inhibitory effect ≦2.5μg phospholipids/ml. Another anionic phospholipid, PS, was lesseffective than PI and POPG. In contrast, the aminophospholipids andsphingolipids DPPC, PE and SM had no significant effect on TNF-α or NOproduction. The major molecular species of PG in humans is POPG, whereasrodent surfactant contains a mixture of disaturated and unsaturated PG.The inventors next examined the effect of saturation and acyl chainlength of PGs on the inhibition of LPS induced inflammation. As shown inFIG. 2, disaturated PGs containing two palmitic (16:0), stearic (18:0),or octanoic (8:0) fatty acids failed to antagonize LPS induced TNF-α orNO production. However, PGs with two myristic (14:0) fatty acids were aspotent as POPG as antagonists of LPS. PGs with two lauric (12:0) fattyacids were also modest antagonists of LPS induced cytokine production.Although the reagents were not available to compare different molecularspecies of PI for LPS antagonism, the PIs that were used wereunsaturated with the major form containing 16:0 and 18:2 fatty acids.From the above findings, it was concluded that unsaturated PGs and PIsand selected saturated PGs act as potent antagonists of LPS action uponmacrophages.

POPG Antagonism of LPS Induced Inflammation Occurs in the Context ofSurfactant Phospholipids.

The POPG present in the alveolar compartment is in a lipid richenvironment with concentrations of total phospholipids of 10-15 mg/ml(32), and the inventors examined whether these other lipids caninterfere with LPS antagonism. Two types of experiments were performed.In one set of experiments, POPG was added to organic solvent extracts ofsurfactant using a method that ensured ideal mixing of all components.In this first procedure, the POPG and all other lipids were randomlymixed in each lipid extract and then liposomes were prepared bysonication. In a second set of experiments, vesicles composed ofsurfactant lipids and vesicles composed of POPG were prepared separatelyand then combined. In this latter situation there will be twopopulations of vesicles, one containing randomly mixed surfactant lipidsand a second containing pure POPG. The results presented in FIG. 3Areveal that surface dilution and randomization of POPG within a singlevesicle significantly diminishes the potency of the lipid as anantagonist of LPS action. In order to approximate the activity of POPGalone, the randomized POPG must now constitute nearly 50% of the totallipid present in a surfactant lipid-containing vesicle. In contrast tothe results in FIG. 3A, the data presented in FIG. 3B demonstrate thatadmixture of pure POPG vesicles and randomized surfactant lipid vesicleshas essentially no effect upon the activity of POPG as an LPSantagonist, measured by TNF-α production. This result also indicatesthat the combination of pure POPG vesicles with surfactant lipidvesicles does not result in significant fusion and intermixing of lipidsbetween vesicle bilayers. This important result suggests that theintroduction of POPG vesicles into the surfactant environment of thealveolar compartment of the lung may yield physical forms of the lipidcapable of potently antagonizing LPS action.

POPG Inhibits the Phosphorylation of MARK and IkBα and Expression ofMKP-1.

The inventors next investigated the influence of phospholipids upon theintracellular signaling pathways of LPS-induced TNF-α secretion. Hostcells recognize many specific microbial components through toll-likereceptors that mediate immune responses. On alveolar macrophages, LPSbinds to membrane CD14 and a TLR4-MD2 complex. The signals from TLR4 aretransmitted through MyD88 and TRAF6 (33) to IkBα or mitogen activatedprotein kinases (MAPKs) such as ERK, JNK and p38. These signals regulatetranscription factors and induce proinflammatory cytokine production. Inexperiments summarized in FIG. 4, differentiated U937 cells werestimulated with LPS in the absence or presence of POPG and cell lysateswere electrophoresed and immunoblotted (FIG. 4). Significant increasesin phosphorylated forms of p38, p42ERK and JNK, as well as IkBα weredetected between 15 and 60 mins after LPS treatment. LPS treatment alsoreduced the steady state levels of IkBα due to protein degradation.Treatment of cells with POPG in addition to LPS eliminated thephosphorylation of p38, p42ERK, JNK and IkBα and also abrogated thereduction in the steady state levels of IkBα. In addition to inducingphosphorylation of MAPKs and IkBα, LPS induces synthesis of MKP-1 thatfunctions to turn off MAPKs signaling (34, 35). The POPG treatmentblocked the synthesis of new MKP-1, indicating that the lipid is likelyto act upstream of MAPK activation rather than downstream of the processby induction of MKP-1.

The quantification of western blotting results from multiple experimentsperformed as shown in FIG. 4 is presented in FIG. 5. The POPG treatmentsignificantly reduces p38, ERK, JNK and IkBα phosphorylation in LPSstimulated cells to values nearly equivalent to untreated cells. Theexpression of MKP1 was also reduced to control levels by POPG treatment.In addition, the total amount of IkBα present in the cells remainedconstant when LPS treated cells were also given POPG. This latter resultdemonstrates that POPG prevents degradation of IkBα that occurssubsequent to LPS treatment alone.

The molecular specificity of the POPG action upon MAPKs, IkBα and MKP-1was also examined as shown in FIG. 6. In these experiments POPG wascompared to POPC and DPPG. The results clearly demonstrate theimportance of the contributions from the polar headgroup and the fattyacid substituents of the phospholipid. Whereas POPG potently inhibitedp38, ERK, JNK, and IkBα phosphorylation and MKP-1 protein expression,neither POPC nor DPPG exerted a significant effect on these parameters.The quantification of the data in FIG. 6 is given in FIG. 7, whichsummarizes results from three independent experiments.

The inventors also conducted control experiments to test whether POPGtreatment had a general toxic effect upon U937 cells. Protein synthesiswas measured by determining [³H]-Leucine incorporation intotrichloroacetic acid precipitable material in the presence and absenceof 200 μg/ml POPG. No changes in protein synthesis occurred over a 6 hrperiod in POPG treated U937 cells compared to untreated cells in 3independent experiments. Additional studies were performed to examinewhether POPG pleiotropically inhibited signaling by macrophages. Inthese studies, macrophages were treated with TNF-α (10 ng/ml) and thedegradation of IkBα was measured. POPG treatment of TNF-α stimulatedmacrophages failed to alter IkBα degradation when compared to stimulatedcells without POPG treatment. Collectively, these studies indicate thatthe actions of the anionic lipids upon LPS signaling are specific.

Anionic Phospholipids Antagonize LPS Activation of Human AlveolarMacrophages.

The inventors next examined whether the findings obtained with humantissue culture macrophages and rat alveolar macrophages were alsorelevant to human alveolar macrophages in primary culture. The humanmacrophages were isolated by bronchoalveolar lavage and challenged with10 ng/ml LPS for 6 hr. The inflammatory response was assessed bymeasuring TNF-α production. The results presented in FIG. 8 demonstratethat POPG, dimyristoyl PG or PI markedly attenuate the inflammatoryresponse of freshly isolated human alveolar macrophages to LPS. Incontrast, DPPG and DPPC had no significant effect upon the humanalveolar macrophage response to LPS. These results demonstrate thathuman macrophages residing in the alveolar compartment are susceptibleto having their inflammatory response to LPS greatly attenuated byanionic surfactant phospholipids and the synthetic lipid DMPG.

POPG Inhibits LPS-induced Proinflammatory Cytokine Production in Vivo.

Since POPG was a strong inhibitor of TNF-α and NO production in vitro,the inventors examined if this lipid can inhibit inflammation in vivo.Phospholipid liposomes were formed using a Liposofast™ apparatus.Mixtures of LPS and phospholipids were sprayed into the trachea of miceusing a MicroSprayer™ positioned at the vocal cords. At 18 h afterstimulation, the lungs of mice were lavaged (FIG. 9) and TNF-α,neutrophil infiltration, and interleukin (IL)-8 equivalents (KC andMIP-2) were measured in the recovered lavage fluid. These threeinflammatory indicators are important prognostic determinants ofALI/ARDS (36). LPS-induced TNF-α was approximately 300 pg/ml and wasunaffected by DPPC instillation. In contrast, POPG, DMPG and PIsignificantly attenuated the TNF-α secretion in the lung. These resultsclearly indicate that the intratrachealy administered POPG and PI canreduce the inflammation in the lung in vivo. These results correlatewell with in vitro results. LPS stimulation also induced theinfiltration of neutrophils. POPG, DMPG and PI, but not DPPC, modestlyattenuated the LPS-induced neutrophil infiltration. Since IL-8 has notbeen identified in mice, the inventors measured KC and MIP-2 as thefunctional homologues of IL-8 (37). DMPG, PI and especially POPGattenuated the KC and MIP-2 secretion in BALF. These findings suggestthat the antagonistic phospholipids will inhibit the secretion of IL-8from human alveolar macrophages in vivo. These results reveal a highpotential for POPG or PI to be used as chemotherapeutic agents forLPS-elicited disorders in human lung.

Next, the effect of phospholipids were examined in a sepsis model inmice. LPS (50 μG/200 μl) was intravenously administered to mice via thetail vein and phospholipids were administered intratrachealy at the sametime. Three hours after stimulation, BALF was collected (FIG. 10).Intratrachealy administered POPG, but not DPPC, significantly inhibitedthe TNF-A secretion in BALF, indicating this anionic lipid has ananti-inflammatory effect for sepsis originating outside the lung. POPGadministered via the trachea, effectively inhibited the infiltration ofneutrophils in BALF compared to DPPC. POPG also significantly attenuatedthe KC and MIP-2 levels in BALF although the magnitude of this effectwas not very large. These results further indicate that POPG may beuseful for treating disorders such as ALI and ARDS in the lung caused bysepsis.

POPG Blocks the Binding of BODIPY-LPS to Macrophages

The results described earlier in this Example in FIGS. 4 and 5demonstrated that the antagonistic phospholipids acted upstream ofkinases involved in LPS signaling. One possible site of action for thephospholipids is at the cell surface. In the experiments shown in FIG.11A, RAW264.7 cells were incubated with fluorescent LPS at 0° C. andquantified the binding by flow cytometry. Untreated macrophages give anautofluorescent profile with a mean fluorescence intensity (MFI) of 3.Incubation of macrophages with BODIPY-LPS produces a shift in MFI to 9,indicating binding of the fluorescent ligand to the cells. Treatment ofthe cells with PI or POPG blocks the binding of BODIPY-LPS to thesurface of the macrophages resulting in almost no increase in MFI. Asummary of the findings with other phospholipids is given in FIG. 9B.The data are expressed as the MFI ratio (treated:untreated control). Theresults show the inhibition of BODIPY-LPS binding to the RAW cells isboth phospholipid headgroup and fatty acid specific. Thus, unsaturatedPI and PG antagonize LPS binding to macrophages whereas long chainsaturated PG and either saturated or unsaturated species of PC arewithout effect.

References for Example 1

1. O'Brien, A. D., Rosenstreich, D. L., Scher, I., Campbell, G. H.,MacDermott, R. P., Formal, S. B. 1980. Genetic control of susceptibilityto Salmonella typhimurium in mice: role of the LPS gene. J Immunol124:20.

2. Ulevitch, R. J., Tobias, P. S. 1995. Receptor-dependent mechanisms ofcell stimulation by bacterial endotoxin. Annu Rev Immunol 13:437.

3. Wright, S. D., Ramos, R. A., Tobias, P. S., Ulevitch, R. J.,Mathison, J. C. 1990. CD14, a receptor for complexes oflipopolysaccharide (LPS) and LPS binding protein. Science 249:1431.

4. Nagai, Y., Akashi, S., Nagafuku, M., Ogata, M., Iwakura, Y., Akira,S., Kitamura, T., Kosugi, A., Kimoto, M., Miyake, K. 2002. Essentialrole of MD-2 in LPS responsiveness and TLR4 distribution. Nat Immunol3:667.

5. Poltorak A., H. X., Smirnova I., Liu M. Y., Van Huffel C., Du X.,Birdwell D., Alejos E., Silva M., Galanos C., Freudenberg M.,Ricciardi-Castagnoli P., Layton B., Beutler B. 1998. Defective LPSsignaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene.Science 282:2085.

6. Takeda, K., Kaisho, T., Akira, S. 2003. Toll-like receptors. Annu RevImmunol 21:335.

7. Medzhitov, R. 2001. Toll-like receptors and innate immunity. Nat RevImmunol 1:135.

8. Barton, G. M., Medzhitov, R. 2003. Toll-like receptor signalingpathways. Science 300:1524.

9. Pattle, R. E. 1955. Properties, function and origin of the alveolarlining layer. Nature 175:1125.

10. Clements, J. A. 1957. Surface tension of lung extracts. Proc Soc ExpBiol Med 95:170.

11. King, R. J., D. J. Klass, E. G. Gikas, and J. A. Clements. 1973.Isolation of apoproteins from canine surface active material. Am JPhysiol 224:788.

12. Kuroki, Y., and D. R. Voelker. 1994. Pulmonary surfactant proteins.J. Biol. Chem. 269:25943.

13. Sano, H., Kuroki, Y. 2005. The lung collectins, SP-A and SP-D,modulate pulmonary innate immunity. Mol Immunol 42:279.

14. Lawson, P. R., Reid, K. B. 2000. The roles of surfactant proteins Aand D in innate immunity. Immunol Rev 173:66.

15. Sano, K., H. Sohma, T. Muta, S.-I. Nomwra, D. R. Voelker, and Y.Kuroki. 1999. Pulmonary surfactant protein A modulates the cellularresponse to smooth and rough lipopolysaccharide by interaction withCD14. J. Immunol. 163:387.

16. Veldhuizen, R., Nag, K., Orgeig, S., Possmayer, F. 1998. The role oflipids in pulmonary surfactant. Biochem Biophys Acta 1408:90.

17. Schmidt, R., Meier, U., Markart, P., Grimminger, F., Velcovsky, H.G., Morr, H., Seeger, W., Gunther, A. 2002. Altered fatty acidcomposition of lung surfactant phospholipids in interstitial lungdisease. Am J Physiol Lung Cell Mol Physiol 283:1079.

18. Wright S M, H. P., Enhorning, G, Strong P, Reid K B, Holgate S T,Djukanovic R, Postle A D. 2000. Altered airway surfactant phospholipidcomposition and reduced lung function in asthma. J Appl Physiol 89:1283.

19. Bochkov, V. N., Kadl, A., Huber, J., Gruber, F., Binder, B. R.,Leitinger, N. 2002. Protective role of phospholipid oxidation productsin endotoxin-induced tissue damage. Nature 419:77.

20. Wu, Y. Z., Medjane, S., Chabot, S., Kubrusly, F. S., Raw, I.,Chignard, M., Touqui, L. 2003. Surfactant protein-A andphosphatidylglycerol suppress type IIA phospholipase A2 synthesis vianuclear factor-kappaB. Am J Respir Crit Care Med 168:692.

21. Hashimoto, M., Asai, Y., Ogawa, T. 2003. Treponemal phospholipidsinhibit innate immune responses induced by pathogen-associated molecularpatterns. J Biol Chem 27:44205.

22. Mueller, M., Brandenburg, K., Dedrick, R., Schromm, A. B., Seydel,U. 2005. Phospholipids inhibit lipopolysaccharide (LPS)-induced cellactivation: a role for LPS-binding protein. J Immunol 172:1091.

23. Atabai, K., Matthay, M. A. 2002. The pulmonary physician in criticalcare. 5: Acute lung injury and the acute respiratory distress syndrome:definitions and epidemiology. Thorax 2002.

24. Rubenfeld, G. D., Caldwell, E., Peabody, E., Weaver, J., Martin, D.P, Neff, M., Stern, E. J., Hudson, L. D. 2005. Incidence and outcomes ofacute lung injury. N Engl J Med 353:1685.

25. Hawgood, S., B. J. Benson, and R. L. Hamilton, Jr. 1985. Effects ofa surfactant-associated protein and calcium ions on the structure andsurface activity of lung surfactant lipids. Biochemistry 24:184.

26. Kuroki, Y., R. J. Mason, and D. R. Voelker. 1988. Pulmonarysurfactant apoprotein A structure and modulation of surfactant secretionby rat alveolar type II cells. J. Biol. Chem. 263:3388.

27. Rouser, G., A. N. Siakatos, and S. Fleischer. 1966. Quantitativeanalysis of phospholipids by thin layer chromatography and phosphorousanalysis of spots. Lipids 1:85.

28. Ding, A. H., Nathan, C. F., Stuehr, D. J. 1988. Release of reactivenitrogen intermediates and reactive oxygen intermediates from mouseperitoneal macrophages. Comparison of activating cytokines and evidencefor independent production. J Immunol 141:2407.

29. Hibi, M., Lin, A., Smeal, T., Minden, A., Karin, M. 1993.Identification of an oncoprotein- and UV-responsive protein kinase thatbinds and potentiates the c-Jun activation domain. Genes Dev 7:2135.

30. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulose sheet:Procedures and some applications. Proc Natl Acad Sci USA 76:4350.

31. Flotte, T. R., Laube, B. L. 2001. Gene therapy in cystic fibrosis.Chest 120(3 Suppl): 124S.

32. Lewis, J. F., and A. H. Jobe. 1993. Surfactant and the adultrespiratory distress syndrome. Am Rev Respir Dis 147:218.

33. Akira, S., Takeda, K. 2004. Toll-like receptor signalling. Nat RevImmunol 4:499.

34. Chen, P., Li, J., Barnes, J., Kokkonen, G. C., Lee, J. C., Liu, Y.2002. Restraint of proinflammatory cytokine biosynthesis bymitogen-activated protein kinase phosphatase-1 inlipopolysaccharide-stimulated macrophages. J Immunol 169:6408.

35. Zhao, Q., Shepherd, E. G., Manson, M. E., Nelin, L. D., Sorokin, A.,Liu, Y. 2005. The role of mitogen-activated protein kinase phosphatase-1in the response of alveolar macrophages to lipopolysaccharide:attenuation of proinflammatory cytokine biosynthesis via feedbackcontrol of p38. J Biol Chem 280:8101.

36. Meduri, G., U, Kohler, G., Headley, S., Tolley, E., Stentz, F.,Postlethwaite, A. 1995. Inflammatory cytokines in the BAL of patientswith ARDS. Persistent elevation over time predicts poor outcome. Chest108:1303.

37. Wuyts, A., Haelens, A., Proost, P., Lenaerts, J. P., Conings, R.,Opdenakker, G., Van Damme, J. 1996. Identification of mouse granulocytechemotactic protein-2 from fibroblasts and epithelial cells. Functionalcomparison with natural KC and macrophage inflammatory protein-2. JImmunol 157:1736.

38. Wright, J. R. 2005. Immunoregulatory functions of surfactantproteins. Nat Rev Immunol 5:58.

39. Nag, K., J. Perez-Gil, M. L. Ruano, L. A. Worthman, J. Stewart, C.Casals, and K. M. Keough. 1998. Phase transitions in films of lungsurfactant at the air-water interface. Biophys J 74:2983.

40. Honda, Y., Tsunematsu, K., Suzuki, A., Akino, T. 1988. Changes inphospholipids in bronchoalveolar lavage fluid of patients withinterstitial lung diseases. Lung 166:293.

41. Saydain, G., Islam, A., Afessa, B., Ryu, J. H., Scott, J. P, Peters,S. G. 2002. Outcome of patients with idiopathic pulmonary fibrosisadmitted to the intensive care unit. Am J Resp Crit Care Med 166:839.

42. Schmidt, R., Meier, U., Yabut-Perez, M., Walmrath, D., Grimminger,F., Seeger, W., Gunther, A. 2001. Alteration of fatty acid profiles indifferent pulmonary surfactant phospholipids in acute respiratorydistress syndrome and severe pneumonia. Am J Respir Crit Care Med163:95.

Example 2

The following experimental results describe the mechanistic basis of thesurfactant lipid antagonism of LPS action. In particular, this exampleshows that CD14 binds POPG and PI with high affinity. The binding ofPOPG to CD14 almost completely inhibits the interaction of the proteinwith LPS. Monoclonal antibodies known to occlude the CD14 binding pocketfor LPS also block the interactions of POPG and PI with CD14. Inaddition to binding CD14, POPG also partially inhibits the interactionsbetween LBP and CD14. The TLR4 associated protein MD-2, which binds LPS,also binds POPG with high affinity. The phospholipid binding by MD-2inhibits its interaction with TLR4. Although the actions of PI aresimilar to POPG, the principal mode of action by PI appears to be byinterference in CD14 function. By comparison, POPG acts at the level ofLBP, CD14 and MD-2 to suppress TLR4 signaling. These findingsdemonstrate a major role for POPG in human and other mammalian pulmonarysurfactants as a suppressor of unwanted inflammatory events in thealveolar compartment of the lung.

Experimental Procedures

Cells and Reagents. LPS (0111:B4) purified from Escherichia coli waspurchased from Sigma-Aldrich (St. Louis, Mo.). Phosphatidylcholine (PC),phosphatidylglycerol (PG), and phosphatidylinositol (PI) in chloroform,were purchased from Avanti Polar Lipids (Alabaster, Ala.). Recombinanthuman CD14, and mouse anti-CD14 monoclonal antibodies, biG2 and biG14,were purchased from Cell Sciences Inc. (Canton, Mass.). Mouse anti-CD14monoclonal antibody MEM-18 was purchased from Exbio (Czech Republic).Mouse anti-H is antibody and HRP-conjugated mouse anti-V5 antibody wereobtained from Invitrogen Life Technologies (Carlsbad, Calif.). MouseIgG₁ isotype control, mouse monoclonal anti-human CD14 antibody, sheepanti-human CD14 polyclonal antibody, recombinant human LBP and goatanti-human LBP antibody were purchased from R&D systems (Minneapolis,Minn.). The macrophage-like cell line U937 (CRL-1593.2) was obtainedfrom American Type Culture Collection (Manassas, Va.). The cells weremaintained in endotoxin-free Roswell Park Memorial Institute (RPMI) 1640medium from Cambrex (East Rutherford, N.J.) with 10% heat-inactivatedbovine growth serum (BGS; Hyclone, Logan, Utah).

Soluble Extracellular Domain of TLR4 and MD-2. A soluble form of theextracellular domain of TLR4 (sTLR4) consists of the putativeextracellular sequence (Met¹-Lys⁶³¹) and a 6 His epitope tag at itsC-terminal end. sTLR4 and MD-2 cDNAs were described previously (10).sTLR4-His was subcloned into pcDNA3.1(+) (Invitrogen Life Technologies).MD-2-V5-His that contains the C-terminal fusion V5 epitope tag and 6 Hisepitope tag was generated by using PCR, and subcloned intopcDNA3.1D/V5-His-TOPO (Invitrogen Life Technologies). A control protein,yeast PstB2-V5-His that contains the C-terminal fusion V5 tag epitopeand 6 His tag epitope was generated by using PCR and subcloned into thebaculovirus vector pVL1392 (16). The epitope tagged cDNA constructs forsTLR4 and MD-2 were subcloned into PVL1392 and in addition to PstB2 wereindependently expressed using a baculovirus-insect cell expressionsystem according to the methods described by O'Reilly et al (17). ThesTLR4 protein and the MD-2 protein were purified using a column ofnickel-nitrilotriacetic acid beads (Qiagen, Valencia, Calif.) by themethod described previously (12).

Induction of TNF-α Secretion. U937 cells were induced to differentiateby incubation in medium containing 10 nM of phorbol myristate acetate(PMA) for 48 h. The cells (1.3×10⁵/well) were placed on 96-well platesand further incubated in the absence of PMA for 24 h in RPMI 1640 mediumcontaining 10% BGS. After the cells were washed with PBS, the indicatedconcentration of phospholipids was preincubated with the cells in RPMIwithout serum for 30 min before adding LPS. The indicated amount of LPSwas then added into the well and incubated for 6 h at 37° C. with 5%CO₂. The culture medium was collected and assayed for TNF-α secretionusing an ELISA kit (Invitrogen).

Binding of CD14 and MD-2 to Phospholipids. Phospholipids (1.25 nmole) in20 μl aliquots of ethanol were pipeted onto 96-well half-area plates(Corning Inc., Corning, N. Y.), and the solvent evaporated using a warmair blower. After nonspecific binding was blocked with 20 mM Tris buffer(pH 7.4) containing 0.15 M NaCl, 5 mM CaCl₂ or 2 mM EGTA, and 5%(wt/vol) BSA (buffer A), various concentrations of human CD14 or MD-2 in25 μl of buffer A were added and incubated at 37° C. for 1 h. The wellswere then washed with 20 mM Tris buffer (pH 7.4) containing 0.15 M NaCland 5 mM CaCl₂ or 2 mM EGTA (buffer B), and 1 μg/ml anti-human CD14 IgGor anti-His antibody (50 μl/well) in buffer A was added and incubatedovernight at 4° C., followed by the incubation with horseradishperoxidase (HRP)-labeled anti-mouse IgG (1:5000) for 1 h. After washingthe wells with buffer B, the peroxidase reaction was finally performedusing o-phenylenediamine as a substrate. The binding of CD14 or MD-2 tophospholipids was detected by measuring absorbance at 490 nm.

Binding of LPS to CD14. LPS (2 μg) in 20 μl aliquots of ethanol waspipeted onto a 96-well plate, and the solvent evaporated using a warmair dryer. After the nonspecific binding was blocked with buffer A,mixtures of CD14 (1 μg/ml) and phospholipid liposomes (20 μg/ml) inbuffer A, which were preincubated at 37° C. for 1 h, were added andfurther incubated at 37° C. for 1 h. The amount of bound CD14 wasdetected using the method described above.

Phospholipid Competition for LBP-CD14 Binding and MD-2-sTLR4 Binding.CD14 (2 μg) or sTLR4 (100 ng) in aliquots of 20 μl of buffer B werepipeted onto a 96-well plate, and the solvent evaporated using a warmair dryer. After the nonspecific binding was blocked with buffer A, themixtures of LBP and phospholipid liposomes or the mixture of MD-2 (1μg/ml) and phospholipid liposomes in buffer A were added and incubatedat 37° C. for 1 h. The amount of bound LBP or MD-2 was detected usingspecific antibodies.

Statistical Analysis. All results were expressed as mean±S.E. ANOVA wasused to determine the levels of difference between all groups. Groupswere compared by unpaired two-tailed t-test. The p-value forsignificance was set at 0.05.

Results

CD14 Binds POPG and PI in a Concentration Dependent Manner.

LPS mainly binds to CD14 on cell surfaces, and is subsequentlytransferred to an MD-2/TLR4 complex on the same membrane to initiatesignaling. We investigated whether CD14 is a target for phospholipidinteraction that antagonizes LPS action. The anionic surfactant lipidsPOPG and PI, adsorbed as a solid phase to microtiter wells, stronglybound to CD14 in a concentration dependent manner (FIG. 12). The bindingof CD14 to the zwitterionic surfactant lipid, DPPC, was significantlyless (approximately 30%) than the levels of POPG binding. Theseinteractions were not attenuated by EGTA indicating that CaCl₂ was notrequired for the binding. These results demonstrate that POPG and PI candirectly bind CD14 and these interactions are of higher affinity thatthose with DPPC. These binding interactions for the anionicphospholipids are consistent with the effect of these same lipids uponinflammatory mediator production, and fluorescent LPS binding tomacrophages, described in the accompanying paper. The molecular speciesof PG were also evaluated for their direct binding interactions withCD14. POPG and DMPG exhibited the strongest direct binding interactionswith CD14. However, CD14 also bound to DPPG to nearly the same extent asPOPG and DMPG (FIG. 13A). These results clearly indicate thatanti-inflammatory anionic phospholipids can bind strongly to CD14.However, some lipids without demonstrable anti-inflammatory effect alsowill directly bind CD14.

POPG Blocks the Interaction of CD14 with LPS

Another method to evaluate CD14-lipid interaction is to performcompetition experiments in which CD14 binding to LPS is subjected tocompetition using phospholipids. This series of experiments is describedin FIG. 13B. Two lipids that function as potent LPS antagonists in vitroand vivo, DMPG and POPG, are the most effective inhibitors of CD14binding to solid phase LPS. DPPG, which is inactive as an LPSantagonist, weakly competes for CD14 binding to LPS. These latterfindings are consistent with earlier findings about PG antagonism of LPSactivation of macrophages. Paradoxically, PI, which is a potent LPSantagonist fails to compete for CD14 binding to solid phase LPS. Theselatter results strongly suggest that PI and POPG do not act by identicalmechanisms in producing LPS antagonism.

POPG and PI Bind to CD14 at the LPS Binding Site.

CD14 has four LPS binding sites located at the N-terminus of the protein(18). Monoclonal antibodies biG14 and MEM-18 recognize aa39-44 andaa57-64, respectively, that constitute part of the LPS binding site. ThebiG14 and MEM-18 antibodies are also proven inhibitors of LPS-binding toCD14. Another antibody, biG2, recognizes aa147-152, which is not part ofthe LPS binding site, and biG2 ligation does not inhibit LPS-binding toCD14. The epitope for another antibody, RDIg, has not been determined,but appears to recognize a site distinct from that used for LPS binding.Recent solution of the crystal structure of mouse CD14 at a resolutionof 2.5 Å provides evidence that LPS binds a defined pocket in theprotein (19). The biG14 and MEM-18 binding sites are close to the pocketand predicted to stearically occlude LPS binding. In contrast, the biG2site when ligated by antibody should not interfere with LPS binding. Theinventors examined the action of the above-described monoclonalantibodies to determine the relationship between the LPS binding siteand the anionic phospholipid binding site on CD14.

In these experiments, the CD14 was preincubated with specific monoclonalantibodies and the effect of this interaction upon the recognition ofsolid phase phospholipid by CD14 was measured. The CD14 bound to thesolid phase was detected using anti-CD14 polyclonal antibody. As shownin FIG. 14A, the monoclonal antibodies biG14 and MEM-18 significantlyreduced the CD14 binding to solid phase POPG by 40-60%, whereas otherantibodies that did not recognize the LPS binding site (mouse IG, RDIgand biG2), failed to significantly alter CD14 recognition of POPG. Theaddition of biG14 and MEM-18 antibodies together gave slightly higherinhibition of CD14 binding to lipid than either antibody alone. Nearlyidentical results were obtained when PI was used as the solid phaseligand as shown in FIG. 14B, with the monoclonal antibodies inhibitingthe binding reaction by 40-70%. Interestingly, none of the antibodiestested inhibited the binding of CD14 to DPPG (data not shown). Thus, thesite of interaction between CD14 and POPG and PI, is different from thesite of interaction with DPPG. In FIG. 14C the inventors conductedcontrol experiments with solid phase CD14 to show that ligation of theprotein by RDIg, biG14, MEM-18 and biG2 does not attenuate the bindingof anti-CD14 polyclonal antibody. Thus the loss of polyclonal antibodydetection of CD14 reflects a reduction in interaction of the proteinwith phospholipids, and is not due to monoclonal antibody interferencewith polyclonal antibody recognition.

Anionic Surfactant Phospholipids Inhibit LPS-induced TNF-α Production inthe Absence of LBP.

LBP is a serum LPS binding protein that facilitates the interaction ofLPS with CD14. As a component situated upstream of TLR4 signaltransduction, LBP constitutes another potential target for anioniclipids. The inventors next investigated whether surfactant phospholipidswere associated with LBP action. When U937 macrophages were stimulatedwith LPS in serum free media, LPS-induced synthesis and secretion ofTNF-A was still inhibited by POPG, DMPG, and PI (FIG. 15A). Theseresults demonstrate that anionic phospholipids can work as LPSantagonists in the absence of LBP. In situations without LBP, thepotency of POPG and PI is modestly diminished, but the potency of DMPGas an LPS antagonist continues to remain high. Although theseexperiments demonstrate that LBP is not required for lipid antagonism ofLPS action, they do not address the question of whether directinteractions between LBP and CD14 can be disrupted by anionic lipids. Toinvestigate this latter issue, solid phase CD14 were prepared and theactivity of anionic lipids as competitors for LBP binding was examinedThe findings presented in FIG. 15B demonstrate that POPG cansignificantly reduce the interaction of LBP with CD14 by approximately40%. This inhibitory action of POPG was not exhibited by any othermolecular species of PG tested or PI or DPPC. These findings show thatPOPG acts at more than one step in altering host recognition of LPS anddistinguishes the action of this lipid from the other antagonisticlipids.

POPG Binds to MD-2 and Blocks the Interaction of MD-2 with sTLR4.

TLR4 requires MD-2 for CD14-dependent cellular response to LPS. It isknown that LPS binds to CD14 and MD-2, but not TLR4. Next, the inventorsexamined whether phospholipids directly interact with MD-2 or TLR4.Recombinant MD-2, sTLR4 and the yeast protein PstB2, all with a (His)₆epitope tag, were expressed using the baculovirus-insect cell expressionsystem. Solid phase POPG, strongly bound to MD-2, but not sTLR4 or thecontrol epitope tagged protein PstB2 (FIG. 16A). The lipid recognitionspecificity of MD-2 was evaluated using PI, two molecular species of PCand three molecular species of PG (FIG. 16B). Relative to POPG, onlyDMPG showed significant binding (ca 50% of the POPG value) to MD-2.Neither saturated nor unsaturated PC, nor unsaturated PI, nor DPPGshowed any significant binding to MD-2.

The inventors next probed the influence of lipids upon the interactionsbetween MD-2 and TLR4. The extracellular domain of TLR4 was adsorbedonto microtiter wells, and the direct binding of MD-2 was measured byELISA, using a monoclonal antibody directed against a V5 epitope on theprotein. At low levels of lipid competitor, only POPG interfered withthe MD-2/TLR4 interaction (FIG. 17A) producing 40% inhibition. In FIG.17B the concentration of lipid competitors was varied up to 200 μg/mland only POPG showed any significant inhibition (approximately 75%) ofthe MD-2/TLR4 interaction. The action of POPG as an inhibitor increasedwith increasing concentration of the lipid between 20-200 μg/ml. Theseresults clearly demonstrate that another site of action of POPG occursbetween MD-2 and TLR4. These results further indicate that PI and POPGhave non-identical mechanisms of interaction with the innate immunesystem that result in suppression of inflammation.

References for Example 2

-   1. O'Brien, A. D., Rosenstreich, D. L., Scher, I., Campbell, G. H.,    MacDermott, R. P., Formal, S. B. 1980. Genetic control of    susceptibility to Salmonella typhimurium in mice: role of the LPS    gene. J Immunol 124:20.-   2. Ulevitch, R. J., Tobias, P. S. 1995. Receptor-dependent    mechanisms of cell stimulation by bacterial endotoxin. Annu Rev    Immunol 13:437.-   3. Miller, S. I., R. K. Ernst, and M. W. Bader. 2005. LPS, TLR4 and    infectious disease diversity. Nat Rev Microbiol 3:36.-   4. Clements, J. A. 1957. Surface tension of lung extracts. Proc Soc    Exp Biol Med 95:170.-   5. Wright, S. D., Ramos, R. A., Tobias, P. S., Ulevitch, R. J.,    Mathison, J. C. 1990. CD14, a receptor for complexes of    lipopolysaccharide (LPS) and LPS binding protein. Science 249:1431.-   6. Poltorak A., H. X., Smirnova I., Liu M. Y., Van Huffel C., Du X.,    Birdwell D., Alejos E., Silva M., Galanos C., Freudenberg M.,    Ricciardi-Castagnoli P., Layton B., Beutler B. 1998. Defective LPS    signaling in C3H/HeJ and C57BL/10 ScCr mice: mutations in Tlr4 gene.    Science 282:2085.-   7. Nagai, Y., Akashi, S., Nagafuku, M., Ogata, M., Iwakura, Y.,    Akira, S., Kitamura, T., Kosugi, A., Kimoto, M., Miyake, K. 2002.    Essential role of MD-2 in LPS responsiveness and TLR4 distribution.    Nat Immunol 3:667.-   8. Shimazu, R., S. Akashi, H. Ogata, Y. Nagai, K. Fukudome, K.    Miyake, and M. Kimoto. 1999. MD-2, a molecule that confers    lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med    189:1777.-   9. Viriyakosol, S., P. S. Tobias, R. L. Kitchens, and T. N.    Kirkland. 2001. MD-2 binds to bacterial lipopolysaccharide. J Biol    Chem 276:38044.-   10. Hyakushima, N., H. Mitsuzawa, C. Nishitani, H. Sano, K.    Kuronuma, M. Konishi, T. Himi, K. Miyake, and Y. Kuroki. 2004.    Interaction of soluble form of recombinant extracellular TLR4 domain    with MD-2 enables lipopolysaccharide binding and attenuates    TLR4-mediated signaling. J Immunol 173:6949.-   11. Gioannini, T. L., A. Teghanemt, D. Zhang, N. P. Coussens, W.    Dockstader, S. Ramaswamy, and J. P. Weiss. 2004. Isolation of an    endotoxin-MD-2 complex that produces Toll-like receptor 4-dependent    cell activation at picomolar concentrations. Proc Natl Acad Sci USA    101:4186.-   12. Sano, H., H. Chiba, D. Iwaki, H. Sohma, D. R. Voelker, and Y.    Kuroki. 2000. Surfactant proteins A and D bind CD14 by different    mechanisms. J Biol Chem 275:22442.-   13. Sano, K., H. Sohma, T. Muta, S.-I. Nomwra, D. R. Voelker, and Y.    Kuroki. 1999. Pulmonary surfactant protein A modulates the cellular    response to smooth and rough lipopolysaccharide by interaction with    CD14. J. Immunol. 163:387.-   14. Shepherd, V. L. 2002. Distinct roles for lung collectins in    pulmonary host defense. Am J Respir Cell Mol Biol 26:257.-   15. Hashimoto, M., Asai, Y., Ogawa, T. 2003. Treponemal    phospholipids inhibit innate immune responses induced by    pathogen-associated molecular patterns. J Biol Chem 278:44205.-   16. Wu, W. I., S. Routt, V. A. Bankaitis, and D. R. Voelker. 2000. A    new gene involved in the transport-dependent metabolism of    phosphatidylserine, PSTB2/PDR17, shares sequence similarity with the    gene encoding the phosphatidylinositol/phosphatidylcholine transfer    protein, SEC14. J. Biol. Chem. 275:14446.-   17. O'Reilly, D. R., L. K. Miller, and V. A. Luckow. 1992. In    Baculovirus Expression Vectors. A Laboratory Manual. W.H. Freeman    and Company, New York, p. 109.-   18. Viriyakosol, S., Kirkland, T. N. 1995. A region of human CD14    required for lipopolysaccharide binding. J Biol Chem 270:361.-   19. Kim, J. I., Lee, C. J., Jin, M. S, Lee, C. H., Paik, S. G., Lee,    H., Lee, J. O. 2005. Crystal structure of CD14 and its implications    for lipopolysaccharide signaling. J Biol Chem 280:11347.-   20. Cunningham, M. D., R. A. Shapiro, C. Seachord, K. Ratcliffe, L.    Cassiano, and R. P. Darveau. 2000. CD14 employs hydrophilic regions    to “capture” lipopolysaccharides. J Immunol 164:3255.-   21. Mueller, M., Brandenburg, K., Dedrick, R., Schromm, A. B.,    Seydel, U. 2005. Phospholipids inhibit lipopolysaccharide    (LPS)-induced cell activation: a role for LPS-binding protein. J    Immunol 172:1091.-   22. Inohara, N., and G. Nunez. 2002. ML—a conserved domain involved    in innate immunity and lipid metabolism. Trends Biochem Sci 27:219.-   23. Rubenfeld, G. D., Caldwell, E., Peabody, E., Weaver, J.,    Martin, D. P, Neff, M., Stern, E. J., Hudson, L. D. 2005. Incidence    and outcomes of acute lung injury. N Engl J Med 353:1685.

Example 3

This example demonstrates that various anionic surfactant lipids inhibitthe activity of toll-like receptors.

FIG. 18 illustrates the results described below, and demonstrates thatunsaturated phosphatidylglycerol (POPG) antagonizes the activation ofmultiple Toll-like receptors (TLRs) in RAW 264.7 cells. In thisexperiment, multiple TLRs were stimulated with defined TLR agonists for24 h and the TNFα production was measured. In each case, the maximalstimulation by agonist is set at 100% and the antagonism by POPG isexpressed as the relative % response. The Controls consist of notreatment, or treatment with POPG in the absence of agonist asindicated. The agonists for the TLRs consist of Pam3Cys for TLR1/2,Mycoplasma pneumoniae membranes as an agonist for TLR2, mycoplasmaderived MALP2 as an agonist for TLR 2/6, double stranded RNA (poly IC)for TLR3, Gram-negative lipopolysaccharide for TLR4, and CpG rich DNA asan agonist for TLR9. The TLR4 panel also contains the antagonistpolymyxin B (PB) for comparison with POPG. The TLR9 agonist serves as anegative control to show that the action of POPG is not non-specific forantagonism of all TLR signaling. As shown in FIG. 18, POPG inhibits theactivity of TLR1, TLR2, TLR3, TLR4, and TLR6, but not TLR9.

FIG. 19 illustrates the results of the experiment described below, anddemonstrates that unsaturated phosphatidylglycerol (POPG) andphosphatidylinositol (PI) antagonize the action of multiple Toll-likereceptors (TLRs) on BEAS2B epithelial cells. In this experiment,multiple TLRs were stimulated with defined TLR agonists for 48 h, andthe IL-8 production was measured. In each case, the maximal stimulationby agonist is set at 100% and the antagonism by POPG or PI is expressedas the relative % response. The Controls consist of no treatment, ortreatment with POPG, or phosphatidylcholine (POPC), orphosphatidylinositol (PI), in the absence of agonist as indicated. Theagonists for the TLRs consist of Pam3Cys for TLR1/2, Mycoplasmapneumoniae membranes as an agonist for TLR2, Mycoplasma fermentansderived MALP2 as an agonist for TLR 2/6, double stranded RNA (poly IC)for TLR3, and bacterial flagellin (Fla) as an agonist for TLR5. The TLR5agonist serves as a negative control to show that the action of POPG isnot non-specific for antagonism of all TLR signaling. As shown in FIG.19, POPG and PI inhibit the activity of TLR1, TLR2, TLR3, and TLR6, butnot TLR5.

FIG. 20 illustrates the results of the experiment described below, andshows that unsaturated phosphatidylglycerol (POPG) antagonizes theaction of Toll-like receptor 3 on primary normal human bronchialepithelial (NHBE) cells. In this experiment, NHBE cells were stimulatedwith double stranded RNA (polyIC) and the IL-8 production was measuredafter an interval of 24 hr. The maximal stimulation by agonist is set at100% and the antagonism by POPG is expressed as the relative % response.The Controls consist of no treatment, or treatment with POPG, in theabsence of agonist as indicated. As shown in FIG. 20, POPG inhibits theactivity of TLR3.

FIG. 21 illustrates the results of the experiment described below, anddemonstrates that unsaturated phosphatidylglycerol (POPG) antagonizesthe action of multiple Toll-like receptors (TLRs) on primary humanneutrophils. In this experiment, multiple TLRs were stimulated withdefined TLR agonists for 5-24 h as indicated, and the IL-8 productionwas measured. In each case the maximal stimulation by agonist is set at100% and the antagonism by POPG is expressed as the relative % response.The Controls consist of no treatment, or treatment with POPG, in theabsence of agonist as indicated. The agonists for the TLRs consist ofPam3Cys for TLR1/2, double stranded RNA (poly IC) for TLR3,Gram-negative lipopolysaccharide (LPS) for TLR4, and single stranded RNA(polyU) for TLR7/8. As shown in FIG. 21, POPG inhibits the activity ofTLR1, TLR2, TLR3, TLR4, TLR7 and TLR8.

Example 4

This example demonstrates that unsaturated phosphatidyl glycerol (PG),such as POPG, inhibit respiratory syncytial virus (RSV) infection.

Referring to FIG. 22, this experiment demonstrates that unsaturatedphosphatidylglycerol (POPG) inhibits IL-6 and IL-8 production by BEAS2Band normal human bronchial epithelial (NHBE) challenged by infectionwith Respiratory Syncytial Virus (RSV). Monolayers of BEAS2B and NHBEcells were infected with RSV at a multiplicity of 3 for 48 h. Infectionswere performed on cells in either the absence or the presence of POPG(200 ug/ml). Media were harvested 48 h after viral challenge and assayedfor the presence of IL-6 and IL-8 by ELISA. Controls consisted of noviral challenge in either the presence or absence of POPG as indicated.

Referring to FIG. 23, this experiment shows that unsaturatedphosphatidylglycerol (POPG) prevents the cytopathic effects of RSV uponBEAS2B cells. Monolayers of BEAS2B cells were infected with RSV at amultiplicity of 3 for 72 h. Infections were performed on cells in eitherthe absence or the presence of POPG (200 ug/ml). The cell monolayerswere photographed at a magnification of 200×. Controls consisted of noviral challenge in either the presence or absence of POPG as indicated.

Referring to FIG. 24, this experiment shows that unsaturatedphosphatidylglycerol (POPG) prevents the cytopathic effects of RSV uponNHBE cells. Monolayers of NHBE cells were infected with RSV at amultiplicity of 3 for 72 h. Infections were performed on cells in eitherthe absence or the presence of POPG (200 ug/ml). The cell monolayerswere photographed at a magnification of 200×. Controls consisted of noviral challenge in either the presence or absence of POPG as indicated.

Referring to FIG. 25, this experiment shows that unsaturatedphosphatidylglycerol (POPG) prevents viral replication in BEAS2B andNHBE cells. Monolayers of BEAS2B and NHBE cells were infected with RSVat a multiplicity of 3 for 72 h. Infections were performed on cells ineither the absence or the presence of POPG (200 ug/ml). The cellmonolayers were fixed and stained with goat anti-human RSV antibodyconjugated with horseradish peroxidase. The presence of the antibody wasdetected with diaminobenzamidine. Controls consisted of cell layers notexposed to the virus or exposed to POPG in the absence of virus asindicated.

Referring to FIG. 26, this experiment demonstrates that unsaturatedphosphatidylglycerol (POPG), but not unsaturated phosphatidylcholine(POPC) inhibits cytokine production in BEAS2B and NHBE cells challengedwith RSV. Monolayers of BEAS2B and NHBE cells were infected with RSV ata multiplicity of 3 for 48 h. Infections were performed on cells ineither the absence or the presence of POPG (200 ug/ml) and POPC (200ug/ml). Media were harvested 48 h after viral challenge and assayed forthe presence of IL-6 and IL-8 by ELISA. Controls consisted of no viralchallenge in either the presence or absence of POPG; or the presence orabsence of POPC, as indicated.

Referring to FIG. 27, this experiment shows that unsaturatedphosphatidylglycerol (POPG), but not unsaturated phosphatidylcholine(POPC), prevents the cytopathic effects of RSV upon BEAS2B cells.Monolayers of BEAS2B cells were infected with RSV at a multiplicity of 3for 72 h. Infections were performed on cells in either the absence (b)or the presence (c,f) of POPG (200 ug/ml) and POPC (200 ug/ml) asindicated. Cells were photographed at a magnification of 200×. Controls(a,d,e) consisted of cell layers either not exposed to virus or exposedto POPG, and POPC in the absence of virus as indicated.

Referring to FIG. 28, this experiment shows that unsaturatedphosphatidylglycerol (POPG), but not unsaturated phosphatidylcholine(POPC), prevents the cytopathic effects of RSV upon NHBE cells.Monolayers of NHBE cells were infected with RSV at a multiplicity of 3for 72 h. Infections were performed on cells in either the absence (b)or the presence (c,f) of POPG (200 ug/ml) and POPC (200 ug/ml) asindicated. Cells were photographed at a magnification of 200×. Controls(a,d,e) consisted of cell layers either not exposed to virus or exposedto POPG, and POPC in the absence of virus as indicated.

Example 5

This example demonstrates that saturated PtdGro does not block theanti-inflammatory effects of SP-A upon macrophages stimulated with LPS,and unsaturated-PtdGro exerts potent anti-inflammatory effects on thesemacrophages.

U937 macrophages were stimulated with 100 ng/ml smooth LPS for 6 hours.The culture medium was harvested and assayed for the presence of TNFα byELISA. Control cultures received no additions. SP-A was added asindicated at 10 μg/ml. Saturated-(16:0/16:0)-PtdGro was added at 20μg/ml. Unsaturated-(18:1/18:1)-PtdGro was added at 20 μg/ml. FIG. 29shows the TNFα levels.

Example 6

This example demonstrates that the inhibitory effect ofphosphatidylglycerols on LPS-induced inflammatory mediator production ismolecular species specific.

PG liposomes were formed by bath-sonication for 30 minutes at roomtemperature. LPS (10 ng/ml) and different concentrations of PG wereadded to monolayer cultures of differentiated U937 cells (left panel) orrat alveolar macrophages (right panel). Media TNF-α measurements wereperformed 6 hours after stimulation. Media NO measurements wereperformed 24 h after stimulation. LPS stimulation without PG was set at100%. The molecular species of PG shown on the graph are: 16:0/16:0,dipalmitoyl-phosphatidylglycerol; 18:0/18:0,distearoyl-phosphatidylglycerol, 16:0/18:1,palmitoyl-oleoyl-phosphatidylglycerol (POPG); and 18:1/18:1,dioleoyl-phosphatidylglycerol. The data shown are the means±S.E. fromthree separate experiments with duplicate samples in each experiment.The results are presented in FIG. 30.

Example 7

This example demonstrates that POPG, DMPG and PI antagonize the effectsof LPS on primary human alveolar macrophages.

Human alveolar macrophages were isolated from healthy volunteer BALF andplated onto a 96-well plate. Two days after plating, 10 ng/ml of LPS and20 μg/ml of phospholipids (POPG, DMPG and PI) were added to monolayercultures of human alveolar macrophages. 6 h after stimulation, mediawere collected and TNF-α production was determined by ELISA. LPSstimulation without phospholipid was set at 100%. The data shown in FIG.31 are the means±S.E. from three separate experiments with duplicatesamples in each experiment. The average TNF-α secretion after LPSstimulation was 30.7±15.1 ng/ml. Significance—**: p<0.01, when comparedwith LPS stimulation in the absence of POPG.

Example 8

This example demonstrates that POPG inhibits activation of RAW 264.7macrophages by multiple TLRs.

Monolayers of RAW 264.7 cells were stimulated with the TLR1/2 agonist,Pam3CysK4 (1 ug/ml); the TLR2 agonist, mycoplasma membrane (0.1 ug/mlprotein); the TLR2/6 agonist MALP2 (0.1 ng/ml); the TLR3 agonist,polyI:C (100 ug/ml); the TLR4 agonist, LPS (10 ng/ml); and the TLR9agonist, oligo CpG (10 ug/ml) for 5 hours in either the absence orpresence of 200 ug/ml POPG, as indicated. The control consists ofuntreated cells. Cultures treated only with POPG and no agonists, arealso shown in each panel. Cells treated with LPS and polymyxin B (1000units) are also shown in the TLR4 panel. Following treatment, the mediumwas harvested, centrifuged to remove non-adherent cells and processedfor detection of sereted TNFα by ELISA. Values shown in FIG. 32 areaverages±SE for 3 independent experiments.

Example 9

This example demonstrates that POPG inhibits activation of primarybronchial epithelial cells by multiple TLRs.

Monolayers of normal human bronchial epithelial cells were stimulatedwith the TLR3 agonist, polyI:C (0.1 ug/ml); the TLR5 agonist, flagellin(10 ng/ml) and the TLR7/8 agonist polyU (100 ug/ml) for 24 h in eitherthe absence or presence of 200 ug/ml POPG, or POPC as indicated.Cultures were also treated with POPG or POPC alone, as additionalcontrols. After 24 h the medium was harvested and centrifuged to removenon-adherent cells, and processed to detect IL-8 production by ELISA.Values shown in FIG. 33 are averages±SE for 3 independent experiments.

Example 10

This example demonstrates that POPG suppresses inflammatory cytokineproduction in BEAS2B, and normal human bronchial epithelial cells;induced by Respiratory Syncytial Virus (RSV).

Monolayers of the BEAS2B cell line, or normal human bronchial epithelialcells (NHBE), were either untreated (CONL), or infected with RSV at amultiplicity of 0.5-1; in either the presence or absence of 200 ug/mlPOPG and POPC, as indicated. Additional control conditions exposed themonolayers to POPG, or POPC alone, as indicated. At 48 h after theinitiation of infection, the medium was harvested and centrifuged toremove non-adherent cells. The supernatants were processed for detectionof either IL-6, or IL-8 by ELISA. Values shown in FIG. 34 are means±SEfor three independent experiments.

Example 11

This example demonstrates that POPG prevents the killing of BEAS2B cellsby RSV.

Monolayers of BEAS2B cells were infected with RSV at a multiplicity of0.5-1, in either the presence or absence of 200 ug/ml POPG, or 200 ug/mlPOPC, as indicated. After 72 h the cultures were subjected tophotomicrography at a magnification of 200×, as shown in FIG. 35.

Example 12

This example demonstrates that POPG prevents the killing of normal humanbronchial epithelial cells by RSV.

Monolayers of normal human bronchial epithelial cells were infected withRSV at a multiplicity of 0.5-1, in either the presence or absence of 200ug/ml POPG, or 200 ug/ml POPC, as indicated. After 72 h the cultureswere subjected to photomicrography at a magnification of 200×, as shownin FIG. 36.

Example 13

This example demonstrates that POPG binds RSV with high affinity andspecificity, and inhibits IL-8 production from epithelial cells in aconcentration-dependent manner.

Solid phase phospholipids (10 ug) were adsorbed to microtiter wells byevaporation of ethanol solvent. The wells were blocked with albumin andexposed to varying concentrations of RSV as shown in the left panel ofFIG. 37. The wells were washed 3 times with phosphate buffered salineand the bound virus was detected by ELISA using polyclonal rabbitanti-RSV.

Cultures of BEAS2B cells were treated with RSV at a multiplicity of 0.5in either the absence or the presence of varying concentrations of POPGas indicated. After 48 hours, the culture supernatants were harvestedand centrifuged, and the IL-8 production was quantified by ELISA. IL-8levels are shown in the right panel of FIG. 37.

Example 14

This example demonstrates that POPG blocks the binding of RSV toepithelial cells.

Suspensions of 3×10⁵ Hep2 cells were treated with RSV at a multiplicityof 50, at 37 C for 10 min, in either the absence of lipid, or thepresence of 200 ug/ml POPG, or POPC, as indicated. The cells were nextshifted to 0 C and washed 3 times with PBS. The cells were thenincubated with monoclonal mouse anti-RSV antibody for 1 h at 0 C in PBScontaining 5% BSA. The unbound antibody was removed by washing the cells3 times with PBS, 5% BSA. Next, the cells were incubated withphycoerythrin conjugated rabbit anti-mouse antibody in PBS, 5% BSA for 2h. Following this incubation the cells were washed 3 times with PBS andfixed overnight with 1% buffered formalin. The fixed cell preparationwas washed 3 times with PBS and subjected to FACScan analysis as shownin the left panel of FIG. 38. The summary of the mean fluorescenceintensity (MFI) for all conditions is shown in the right panel of FIG.38.

Example 15

This example demonstrates that POPG arrests the progression of RSVinfection.

Monolayers of Hep2 cells were subjected to RSV infection at levels thatproduce ca 100 plaques per well in quantitative plaque assays. The viruswas incubated with the cells for 5 hours prior to agar overlay. The agaroverlays contained either no additions, or POPG (200-500 ug/ml) or POPC(500 ug/ml). Panel a of FIG. 39 shows the appearance of plaques after 5days of culture following fixation and staining with neutral red. Panelb of FIG. 39 shows the appearance of the monolayers after fixation andstaining with anti-RSV antibody (IHC), which reveals the presence ofbullseye plaques as a result of RSV treatment alone, or RSV plus POPCtreatment; and the appearance of indefinite minute plaques (not visiblewith neutral red staining), as a result of RSV plus POPG treatment.Panel c of FIG. 39 shows the magnification of individual plaquesresulting from RSV (1), or RSV+POPG (2) treatment, and stained withneutral red; or RSV (3), or RSV+POPG (4) treatment stained by IHC.

Example 16

This example demonstrates the quantification of the arrest of plaqueprogression.

The effects of POPG upon plaque formation as shown in Panel a of FIG. 39were quantified in 3 independent experiments and are presented in thisfigure. The left panel of FIG. 40 provides the numbers of definite, andminute indefinite plaques formed in the absence and presence of POPG(200-500 ug/ml) as indicated. The right panel of FIG. 40 provides thenumbers of definite plaques observed in the absence and presence ofeither POPG or POPC (200-500 ug/ml) as indicated.

Example 17

This example demonstrates that POPG suppresses RSV infection in vivo.

Groups of 8 mice were inoculated with 10⁷ RSV in either the absence(RSV), or presence of 200 ug/ml POPG (RSV+POPG). Control experimentsconsisted of UV inactivated RSV (URV), or saline treatment (CON), orPOPG treatment without virus (POPG). Three days after the inoculations,the animals were killed and the lungs harvested and processed for viralcontent by quantitative plaque assay (left panel of FIG. 41), andhistopathology (right panel of FIG. 41)

Example 18

This example demonstrates that nanodisc POPG suppresses activation ofTLR4 by LPS in macrophages and TLRs 2, 3 and 6 in epithelial cells.

Monolayers of RAW 264.7 cells were untreated (CONL) or challenged with10 ng/ml LPS for 24 h (LPS), in the absence or presence of 200 ug/mlPOPG in the form of liposomes (POPG) or as nanodiscs (nano). Additionalcontrol experiments included treatment of the cells with POPG ornanodisc POPG in the absence of LPS, or treatment with LPS and polymyxinB (PB), as indicated. At 24 h following the LPS challenge, the culturesupernatants were harvested and the production of NO was measured usingthe Greiss reaction. Results are shown in FIG. 42.

Monolayers of BEAS2B cells were challenged with the TLR1/2 agonist,Pam3Cys (25 ug/ml); the TLR 2/6 agonist MALP2 (10 ng/ml); the TLR2agonist mycoplasma membrane (Mem) (1 ug/ml); the TLR3 agonist, poly I:C(0.1 ug/ml); and the TLR5 agonist, flagellin (100 ng/ml). For eachagonist parallel incubations were performed containing 200 ug/ml POPG asliposomes, or 200 ug/ml POPG as nanodiscs as indicated. After 48 h, theculture supernatants were harvested and the secretion of IL-8 wasmeasured using ELISA, as shown in FIG. 43. FIG. 43 also shows thenegative results of nanodisc POPG upon TLR5 activation of the cells.

Example 19

This example demonstrates that nanodisc PG of various species iseffective at preventing cytopathology in cells induced by RSV and thatboth liposome and nanodisc POPG inhibit plaque formation by RSV.

Monolayers of BEAS2B cells were either uninfected (CONL) or challengedwith RSV at a multiplicity of 0.5/cell (RSV). Where indicated in theFIG. 44, RSV challenged cells were also treated with POPC (200 ug/ml) orPOPG (200 ug/ml) liposomes; or POPG nanodiscs (nanoPOPG) atconcentrations ranging from 100-300 ug/ml. At 72 h after infection, thecultures were examined by photomicrography at a magnification of 200×,as shown in FIG. 44. Selected panels and fields are also shown at 400×magnification in FIG. 45.

To demonstrate that lipid inhibition of RSV infection is molecular classspecific, monolayers of BEAS2B cells were either uninfected (CONL), orchallenged with RSV at a multiplicity of 0.5/cell (RSV). Where indicatedin FIG. 46, RSV challenged cells were also treated with nanodisc formsof dipalmitoyl-phosphatidylcholine (nanoDPPC),palmitoyl-oleoyl-phosphatidylcholine (nanoPOPC), ordioleoyl-phosphatidylcholine (nanoDOPC),dipalmitoyl-phosphatidylglycerol (nanoDPPG),dimyristoyl-phosphatidylglycerol (nanoDMPG), ordioleoyl-phosphatidylglycerol (nanoDOPG). At 72 h after infection, thecultures were examined by photomicrography at a magnification of 200×.As shown in FIG. 46, nanodisc PGs, but not PCs, disrupted RSV infectionand cytopathology.

Quantitative viral plaque assays were performed in the absence (RSV) andthe presence of liposomal and nanodisc lipids (RSV+POPG, andRSV+nano-POPG) as indicated in FIG. 47, using the indicated dilutions ofvirus from a stock of 2×10⁷/ml RSV. Plaque formation progressed for 5days, after which the cultures were fixed, and stained with neutral red.The results presented in FIG. 47 indicate that RSV plaque formation isinhibited by liposome POPG and nanodisc POPG.

Quantitative viral plaques assays were performed in the absence (RSV)and presence of liposomal POPG (RSV+POPG). These samples are identicalto those shown for RSV and liposome POPG in FIG. 47, but also show theinhibition of plaque formation by liposomal POPG, with much higher viralchallenges (10⁻², 10⁻³ dilutions). The results presented in FIG. 48indicate that liposome POPG suppresses plaque formation over a 4-log(10,000-fold) range.

Example 20

This example demonstrates that liposome POPG prevents the cytopathologyand the inflammation-induced by influenza virus.

Monolayers of BEAS2B cells were infected with influenza-A virus H3N2(IFA), at the multiplicities of infection (moi) indicated, in either theabsence or presence of 200 ug/ml POPG as indicated. The cells wereexamined for cytopathic effects and cell death at 72 after infection byphotomicrography at 200× magnification, as shown in FIG. 49. Theseresults indicate that POPG prevents cell death induced by influenza Ainfection in BEAS2B cells at 72 h.

Monolayers of BEAS2B cells were either untreated (CONL), or challengedwith influenza-A virus H3N2 (IFA), at multiplicities ranging from 1-10as indicated. Liposomes composed of 200 ug/ml POPG were added 30 minutesprior to viral infection (IFA+POPG), where indicated. An additionalcontrol condition in which cells were treated with POPG and no IFA wasalso conducted. The culture supernatants were harvested at 24, 48 and 72h after infection and processed for IL-8 detection by ELISA, as shown inFIG. 50. The results indicate that POPG suppresses influenza-A inducedIL-8 production in epithelial cells.

Each reference described or cited herein is incorporated herein byreference in its entirety.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

What is claimed is:
 1. A method to inhibit rhinovirus infection orinflammation associated with rhinovirus infection, comprisingadministering to an individual who has, or is at risk of developing saidinfection or inflammation, an amount of a pure preparation of aphosphatidylglycerol, wherein the amount of the phosphatidylglycerol iseffective to inhibit said infection or inflammation, and wherein thephosphatidylglycerol has the following characteristics: a) has ahydrophobic portion; b) has a negatively charged portion; and c) has anuncharged, polar portion.
 2. The method of claim 1, wherein thephosphatidylglycerol is selected from the group consisting of:unsaturated phosphatidylglycerol, saturated short chainphosphatidylglycerol, or a derivative thereof.
 3. The method of claim 1,wherein the infection or inflammation is associated with a toll-likereceptor (TLR) selected from the group consisting of: TLR1, TLR2, TLR3,TLR6, TLR7, TLR8, and TLR10.
 4. The method of claim 1, wherein theindividual has a rhinovirus infection or inflammation associated withTLR8.
 5. The method of claim 1, wherein the individual has a rhinovirusinfection or inflammation associated with TLR3.
 6. The method of claim1, wherein the individual has a rhinovirus infection or inflammationassociated with TLR7.
 7. The method of claim 1, wherein the purepreparation of the phosphatidylglycerol is administered as acomposition.
 8. The method of claim 1, wherein the phosphatidylglycerolis administered to the respiratory tract of the individual.